Neprilysin inhibitors

ABSTRACT

In one aspect, the invention relates to compounds having the formula: 
     
       
         
         
             
             
         
       
     
     where R 1 -R 5  and X are as defined in the specification, or a pharmaceutically acceptable salt thereof. These compounds have neprilysin inhibition activity. In another aspect, the invention relates to pharmaceutical compositions comprising such compounds; methods of using such compounds; and processes and intermediates for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/772,721, filed on Mar. 5, 2013; the entire disclosure of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel compounds havingneprilysin-inhibition activity. The invention also relates topharmaceutical compositions comprising such compounds, processes andintermediates for preparing such compounds and methods of using suchcompounds to treat diseases such as hypertension, heart failure,pulmonary hypertension, and renal disease.

2. State of the Art

Neprilysin (neutral endopeptidase, EC 3.4.24.11) (NEP), is anendothelial membrane bound Zn²⁺ metallopeptidase found in many organsand tissues, including the brain, kidneys, lungs, gastrointestinaltract, heart, and the peripheral vasculature. NEP degrades andinactivates a number of endogenous peptides, such as enkephalins,circulating bradykinin, angiotensin peptides, and natriuretic peptides,the latter of which have several effects including, for example,vasodilation and natriuresis/diuresis, as well as inhibition of cardiachypertrophy and ventricular fibrosis. Thus, NEP plays an important rolein blood pressure homeostasis and cardiovascular health.

NEP inhibitors, such as thiorphan, candoxatril, and candoxatrilat, havebeen studied as potential therapeutics. Compounds that inhibit both NEPand angiotensin-I converting enzyme (ACE) are also known, and includeomapatrilat, gempatrilat, and sampatrilat. Referred to as vasopeptidaseinhibitors, this latter class of compounds is described in Robl et al.(1999) Exp. Opin. Ther. Patents 9(12): 1665-1677.

SUMMARY OF THE INVENTION

The present invention provides novel compounds that have been found topossess neprilysin (NEP) enzyme inhibition activity. Accordingly,compounds of the invention are expected to be useful and advantageous astherapeutic agents for treating conditions such as hypertension andheart failure.

One aspect of the invention relates to a compound of formula I:

where:

R¹ is selected from the group consisting of H; C₁₋₈alkyl optionallysubstituted with one or more fluoro atoms; C₁₋₃alkylene-C₆₋₁₀aryl;C₁₋₃alkylene-C₁₋₉heteroaryl; C₃₋₇cycloalkyl; C₂₋₃alkylene-OH;—[(CH₂)₂O]₁₋₃CH₃; C₁₋₆alkylene-OC(O)R²⁰; C₁₋₆alkylene-NR²¹R²²;—CH₂CH(NH₂)—COOCH₃; C₁₋₆alkylene-C(O)R²³; C₀₋₆alkylenemorpholine;C₁₋₆alkylene-SO₂—C₁₋₆alkyl;

where R²⁰ is selected from the group consisting of C₁₋₆alkyl,—O—C₁₋₆alkyl, C₃₋₇cycloalkyl, —O—C₃₋₇cycloalkyl, phenyl, —O-phenyl,—NR²¹R²², CH(R²⁵)—NH₂, —CH(R²⁵)—NHC(O)O—C₁₋₆alkyl, and—CH(NH₂)CH₂COOCH₃; and R²¹ and R²² are independently selected from thegroup consisting of H, C₁₋₆alkyl, and benzyl; or R²¹ and R²² are takentogether as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—; R²³ isselected from the group consisting of —O—C₁₋₆alkyl, —O-benzyl, and—NR²¹R²²; R²⁴ is C₁₋₆alkyl or C₀₋₆alkylene-C₆₋₁₀aryl; and R²⁵ is H,—CH₃, —CH(CH₃)₂, phenyl, or benzyl;

R² is —OH, —CH₂OH, or —CH₂—O—C₁₋₆alkyl; and R³ is H or —CH₃;

R⁴ and R⁵ are independently selected from the group consisting ofhydrogen, halo, —OH, —CH₃, —OCH₃, —CN, and —CF₃;

X is H, —C(O)—R⁶, —C(O)—NR⁷R⁸, —C(O)—NR⁹—NR¹⁰R¹¹,—C(O)—NR¹²—NR¹³—C(O)—R¹⁴, or —CH(R¹⁵)—OR¹⁶;

R⁶ is C₁₋₆alkyl, C₁₋₆alkylene-O—C₁₋₆alkyl, C₀₋₁₀aryl, benzyl, orC₁₋₉heteroaryl;

R⁷ is H, —OH, or C₁₋₆alkyl;

R⁸ is C₁₋₆alkyl; —O—C₁₋₆alkyl; C₅₋₆cycloalkyl; C₀₋₁₀aryl; —O—C₆₋₁₀aryl;—O-benzyl; pyridine optionally substituted with halo, —OH, C₁₋₆alkyl, or—O—C₁₋₆alkyl; morpholine; or isoxazolidinone; or R⁷ and R⁸ are takentogether to form a ring selected from the group consisting of:

where a is 1 and R²⁶ is —OH, or a is 2 and each R²⁶ is independentlyhalo or —C₁₋₃alkylene-OH; R²⁷ is —C₁₋₃alkylene-OH, —C(O)NH₂, or —SO₂CH₃;b is 0, or b is 1 and R²⁸ is —C₁₋₃alkylene-OH, or b is 2 and each R²⁸ isC₁₋₆alkyl; R²⁹ is halo, —COOH, —OH, —C₁₋₃alkylene-OH, —CH₂O—CH₃, —CONH₂,—CN, or pyridine; R³⁰ is C₁₋₆alkyl or C₃₋₇cycloalkyl; R³¹ is —OH or—C₁₋₃alkylene-OH; R³² is halo; C₁₋₆alkyl; C₂₋₄alkylene-O—C₁₋₆alkyl;—C(O)O—C₁₋₆alkyl; —C(O)N(CH₃)₂; pyridine; —SO₂CH₃; —C(O)-furan; orphenyl substituted with halo, —O—C₁₋₆alkyl, or —CN; and R³³ is H, —OH,—O—C₁₋₆alkyl or —O—C₆₋₁₀aryl;

R⁹ is H or C₁₋₆alkyl;

R¹⁶ is H or C₁₋₆alkyl;

R¹¹ is C₁₋₆alkyl; C₁₋₉heteroaryl optionally substituted with halo, —OH,C₁₋₆alkyl, or —O—C₁₋₆alkyl; dihydroimidazole; or phenyl optionallysubstituted with one or two groups selected from the group consisting ofhalo, C₁₋₆alkyl, —O—C₁₋₆alkyl, and —NO₂;

R¹² is H or C₁₋₆alkyl;

R¹³ is H or C₁₋₆alkyl;

R¹⁴ is —O-benzyl; pyridine optionally substituted with halo, —OH,C₁₋₆alkyl, or —O—C₁₋₆alkyl; furan; or phenyl substituted with halo, —OH,—O—C₁₋₆alkyl, or —NO₂;

R¹⁵ H or C₁₋₆alkyl;

R¹⁶ is H, C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃, C₁₋₉heteroaryl, benzyl, orC₆₋₁₀aryl optionally substituted with —OH or —OCH₃; or R¹⁵ and R¹⁶ aretaken together to form —(CH₂)₄—;

or a pharmaceutically acceptable salt thereof.

Another aspect of the invention relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a compound of theinvention. Such compositions may optionally contain other therapeuticagents. Accordingly, in yet another aspect of the invention, apharmaceutical composition comprises a compound of the invention as thefirst therapeutic agent, one or more secondary therapeutic agent, and apharmaceutically acceptable carrier. Another aspect of the inventionrelates to a combination of active agents, comprising a compound of theinvention and a second therapeutic agent. The compound of the inventioncan be formulated together or separately from the additional agent(s).When formulated separately, a pharmaceutically acceptable carrier may beincluded with the additional agent(s). Thus, yet another aspect of theinvention relates to a combination of pharmaceutical compositions, thecombination comprising: a first pharmaceutical composition comprising acompound of the invention and a first pharmaceutically acceptablecarrier; and a second pharmaceutical composition comprising a secondtherapeutic agent and a second pharmaceutically acceptable carrier. Inanother aspect, the invention relates to a kit containing suchpharmaceutical compositions, for example where the first and secondpharmaceutical compositions are separate pharmaceutical compositions.

Compounds of the invention possess NEP enzyme inhibition activity, andare therefore expected to be useful as therapeutic agents for treatingpatients suffering from a disease or disorder that is treated byinhibiting the NEP enzyme or by increasing the levels of its peptidesubstrates. Thus, one aspect of the invention relates to a method oftreating patients suffering from a disease or disorder that is treatedby inhibiting the NEP enzyme, comprising administering to a patient atherapeutically effective amount of a compound of the invention. Anotheraspect of the invention relates to a method of treating hypertension,heart failure, or renal disease, comprising administering to a patient atherapeutically effective amount of a compound of the invention. Stillanother aspect of the invention relates to a method for inhibiting a NEPenzyme in a mammal comprising administering to the mammal, a NEPenzyme-inhibiting amount of a compound of the invention.

Since compounds of the invention possess NEP inhibition activity, theyare also useful as research tools. Accordingly, one aspect of theinvention relates to a method of using a compound of the invention as aresearch tool, the method comprising conducting a biological assay usinga compound of the invention. Compounds of the invention can also be usedto evaluate new chemical compounds. Thus another aspect of the inventionrelates to a method of evaluating a test compound in a biological assay,comprising: (a) conducting a biological assay with a test compound toprovide a first assay value; (b) conducting the biological assay with acompound of the invention to provide a second assay value; wherein step(a) is conducted either before, after or concurrently with step (b); and(c) comparing the first assay value from step (a) with the second assayvalue from step (b). Exemplary biological assays include a NEP enzymeinhibition assay. Still another aspect of the invention relates to amethod of studying a biological system or sample comprising a NEPenzyme, the method comprising: (a) contacting the biological system orsample with a compound of the invention; and (b) determining the effectscaused by the compound on the biological system or sample.

Yet another aspect of the invention relates to processes andintermediates useful for preparing compounds of the invention.Accordingly, another aspect of the invention relates to a process ofpreparing compounds of formula I, comprising the step of coupling (i) acompound of formula 1:

with a compound of formula 2 or formula 7 or formula 8 or formic acid:

or (ii) a compound of formula 3:

with a compound of formula 4 or formula 5 or formula 6:

to produce a compound of formula I; where P¹ is H or a carboxyprotecting group selected from methyl, ethyl, t-butyl, benzyl,p-methoxybenzyl, 9-fluorenylmethyl, trimethylsilyl,t-butyldimethylsilyl, and diphenylmethyl; and where the process furthercomprises deprotecting the compound of formula 1 when P¹ is a carboxyprotecting group; and where R²-R¹⁶, are as defined for formula I.Another aspect of the invention relates to a process of preparing apharmaceutically acceptable salt of a compound of formula I, comprisingcontacting a compound of formula I in free acid or base form with apharmaceutically acceptable base or acid. In other aspects, theinvention relates to products prepared by any of the processes describedherein, as well as novel intermediates used in such process.

Yet another aspect of the invention relates to the use of a compound offormula I or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament, especially for the manufacture of amedicament useful for treating hypertension, heart failure, or renaldisease. Another aspect of the invention relates to use of a compound ofthe invention for inhibiting a NEP enzyme in a mammal. Still anotheraspect of the invention relates to the use of a compound of theinvention as a research tool. Other aspects and embodiments of theinvention are disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an,” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, and so forth used herein are tobe understood as being modified in all instances by the term “about,”unless otherwise indicated. Accordingly, the numbers set forth hereinare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each number should at least be construed in lightof the reported significant digits and by applying ordinary roundingtechniques.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms and include, for example,C₁₋₄alkyl, C₁₋₅alkyl, C₂₋₅alkyl, C₁₋₆alkyl, C₂₋₆alkyl, C₁₋₈alkyl, andC₁₋₁₀alkyl. Representative alkyl groups include, by way of example,methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl,n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown preceding the term assubscript. For example, the term “C₁₋₆alkyl” means an alkyl group havingfrom 1 to 6 carbon atoms, and the term “C₃₋₇cycloalkyl” means acycloalkyl group having from 3 to 7 carbon atoms, respectively, wherethe carbon atoms are in any acceptable configuration.

The term “alkylene” means a divalent saturated hydrocarbon group thatmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 0 to 10 carbon atoms and include, forexample, C₀₋₁alkylene, C₀₋₂alkylene, C₀₋₆alkylene, C₁₋₃alkylene,C₁₋₆alkylene, and C₂₋₄alkylene. Representative alkylene groups include,by way of example, —CH₂—, —(CH₂)₂—, —(CH₂)₃—, —CH₂—CH(CH₃)—, —(CH₂)₄—,—CH₂—CH(CH₃)—CH₂—, —(CH₂)₅—, —CH₂—CH(CH₃)—CH₂—CH₂—, and the like. It isunderstood that when the alkylene term include zero carbons such as—C₀₋₁alkylene-, such terms are intended to include the absence of carbonatoms, that is, the alkylene group is not present except for a covalentbond attaching the groups separated by the alkylene term.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or one or more fused rings. Fused ring systemsinclude those that are fully unsaturated (e.g., naphthalene) as well asthose that are partially unsaturated (e.g.,1,2,3,4-tetrahydronaphthalene). Unless otherwise defined, such arylgroups typically contain from 6 to 10 carbon ring atoms and include, forexample, C₆₋₁₀aryl. Representative aryl groups include, by way ofexample, phenyl and naphthalene-1-yl, naphthalene-2-yl, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms and include, for example,C₃₋₅cycloalkyl, C₃₋₆cycloalkyl, C₃₋₇cycloalkyl, and C₅₋₆cycloalkyl.Representative cycloalkyl groups include, by way of example,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “heteroaryl” is intended to mean a monovalent unsaturated(aromatic) heterocycle having a single ring or two fused rings.Monovalent unsaturated heterocycles are also commonly referred to as“heteroaryl” groups. Unless otherwise defined, heteroaryl groupstypically contain from 5 to 10 total ring atoms, of which 1 to 9 arering carbon atoms, and 1 to 4 are ring heteroatoms, and include, forexample, C₁₋₉heteroaryl and C₅₋₉ heteroaryl. Representative heteroarylgroups include, by way of example, pyrrole (e.g., 1H-pyrrole,2H-pyrrole, and 3H-pyrrole), imidazole (e.g., 2-imidazole), furan (e.g.,2-furan and 3-furan), thiophene (e.g., 2-thiophene), triazole (e.g.,1,2,3-triazole and 1,2,4-triazole), pyrazole (e.g., 1H-pyrazole and4H-pyrazole), oxazole (e.g., 2-oxazole), isoxazole (e.g., 3-isoxazole),thiazole (e.g., 2-thiazole and 4-thiazole), and isothiazole (e.g.,3-isothiazole), pyridine (e.g., 2-pyridine, 3-pyridine, and 4-pyridine),pyridylimidazole, pyridyltriazole, pyrazine, pyridazine (e.g.,3-pyridazine), pyrimidine (e.g., 2-pyrimidine), tetrazole, triazine(e.g., 1,3,5-triazine), indole (e.g., 1H-indole), benzofuran,benzothiophene (e.g., benzo[b]thiophene), benzimidazole, benzoxazole,benzothiazole, benzotriazole, quinoline (e.g., 2-quinoline),isoquinoline, quinazoline, quinoxaline and the like.

The term “optionally substituted” means that group in question may beunsubstituted or it may be substituted one or several times, such as 1to 3 times, or 1 to 5 times, or 1 to 8 times. For example, an alkylgroup that is “optionally substituted” with fluoro atoms may beunsubstituted, or it may contain 1, 2, 3, 4, 5, 6, 7, or 8 fluoro atoms.Similarly, a group that is “optionally substituted” with one or twoC₁₋₆alkyl groups, may be unsubstituted, or it may contain one or twoC₁₋₆alkyl groups.

As used herein, the phrase “having the formula” or “having thestructure” is not intended to be limiting and is used in the same waythat the term “comprising” is commonly used. For example, if onestructure is depicted, it is understood that all stereoisomer andtautomer forms are encompassed, unless stated otherwise.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise unacceptable when used in the invention. Forexample, the term “pharmaceutically acceptable carrier” refers to amaterial that can be incorporated into a composition and administered toa patient without causing unacceptable biological effects or interactingin an unacceptable manner with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug administration.

The term “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (for example, salts having acceptable mammalian safetyfor a given dosage regime). However, it is understood that the saltscovered by the invention are not required to be pharmaceuticallyacceptable salts, such as salts of intermediate compounds that are notintended for administration to a patient. Pharmaceutically acceptablesalts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. In addition, when a compound of formula I contains both a basicmoiety, such as an amine, pyridine or imidazole, and an acidic moietysuch as a carboxylic acid or tetrazole, zwitterions may be formed andare included within the term “salt” as used herein. Salts derived frompharmaceutically acceptable inorganic bases include ammonium, calcium,copper, ferric, ferrous, lithium, magnesium, manganic, manganous,potassium, sodium, and zinc salts, and the like. Salts derived frompharmaceutically acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable inorganic acids include salts of boric,carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric orhydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Saltsderived from pharmaceutically acceptable organic acids include salts ofaliphatic hydroxyl acids (for example, citric, gluconic, glycolic,lactic, lactobionic, malic, and tartaric acids), aliphaticmonocarboxylic acids (for example, acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (for example, aspartic andglutamic acids), aromatic carboxylic acids (for example, benzoic,p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylaceticacids), aromatic hydroxyl acids (for example, o-hydroxybenzoic,p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic and3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids(for example, fumaric, maleic, oxalic and succinic acids), glucoronic,mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids(for example, benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic,isethionic, methanesulfonic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic andp-toluenesulfonic acids), xinafoic acid, and the like.

As used herein, the term “prodrug” is generally intended to mean aninactive precursor of a drug that is converted into its active form inthe body under physiological conditions, for example, by normalmetabolic processes. Such compounds may not possess pharmacologicalactivity at NEP, but may be administered orally or parenterally andthereafter metabolized in the body to form compounds that arepharmacologically active at NEP. When orally administered, suchcompounds may also provide a better fraction absorbed (i.e., better pKproperties) for renal delivery, as compared to oral administration ofthe active form. Exemplary prodrugs include esters such asC₁₋₆alkylesters and aryl-C₁₋₆alkylesters. In one embodiment, the activecompound has a free carboxyl and the prodrug is an ester derivativethereof, i.e., the prodrug is an ester such as —C(O)OCH₂CH₃. Such esterprodrugs are then converted by solvolysis or under physiologicalconditions to be the free carboxyl compound. The term “prodrug” is alsointended to include a less active precursor of a drug that is convertedinto a more active form in the body. For example, certain prodrugs maypossess pharmacological activity at NEP, but not necessarily at thedesired level; such compounds are converted in the body into a formhaving the desired level of activity. The term is also intended toinclude certain protected derivatives of compounds of formula I that maybe made prior to a final deprotection stage. Thus, all protectedderivatives and prodrugs of compounds formula I are included within thescope of the invention.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need thereof, thatis, the amount of drug needed to obtain the desired therapeutic effect.For example, a therapeutically effective amount for treatinghypertension is an amount of compound needed to, for example, reduce,suppress, eliminate, or prevent the symptoms of hypertension, or totreat the underlying cause of hypertension. In one embodiment, atherapeutically effective amount is that amount of drug needed to reduceblood pressure or the amount of drug needed to maintain normal bloodpressure. On the other hand, the term “effective amount” means an amountsufficient to obtain a desired result, which may not necessarily be atherapeutic result. For example, when studying a system comprising a NEPenzyme, an “effective amount” may be the amount needed to inhibit theenzyme.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as hypertension) in apatient, such as a mammal (particularly a human) that includes one ormore of the following: (a) preventing the disease or medical conditionfrom occurring, i.e., preventing the reoccurrence of the disease ormedical condition or prophylactic treatment of a patient that ispre-disposed to the disease or medical condition; (b) ameliorating thedisease or medical condition, i.e., eliminating or causing regression ofthe disease or medical condition in a patient; (c) suppressing thedisease or medical condition, i.e., slowing or arresting the developmentof the disease or medical condition in a patient; or (d) alleviating thesymptoms of the disease or medical condition in a patient. For example,the term “treating hypertension” would include preventing hypertensionfrom occurring, ameliorating hypertension, suppressing hypertension, andalleviating the symptoms of hypertension (for example, lowering bloodpressure). The term “patient” is intended to include those mammals, suchas humans, that are in need of treatment or disease prevention or thatare presently being treated for disease prevention or treatment of aspecific disease or medical condition, as well as test subjects in whichcompounds of the invention are being evaluated or being used in anassay, for example an animal model.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

In one aspect, the invention relates to compounds of formula I:

or a pharmaceutically acceptable salt thereof.

As used herein, the term “compound of the invention” includes allcompounds encompassed by formula I such as the species embodied informulas II-VII and a-f, and combinations thereof. In addition, thecompounds of the invention may also contain several basic or acidicgroups (for example, amino or carboxyl groups) and therefore, suchcompounds can exist as a free base, free acid, or in various salt forms.All such salt forms are included within the scope of the invention.Furthermore, the compounds of the invention may also exist as prodrugs.Accordingly, those skilled in the art will recognize that reference to acompound herein, for example, reference to a “compound of the invention”or a “compound of formula I” includes a compound of formula I as well aspharmaceutically acceptable salts and prodrugs of that compound unlessotherwise indicated. Further, the term “or a pharmaceutically acceptablesalt and/or prodrug thereof” is intended to include all permutations ofsalts and prodrugs, such as a pharmaceutically acceptable salt of aprodrug. Furthermore, solvates of compounds of formula I are includedwithin the scope of this invention.

The compounds of formula I may contain one or more chiral centers andtherefore, these compounds may be prepared and used in variousstereoisomeric forms. Accordingly, the invention also relates to racemicmixtures, pure stereoisomers (e.g., enantiomers and diastereoisomers),stereoisomer-enriched mixtures, and the like unless otherwise indicated.When a chemical structure is depicted herein without anystereochemistry, it is understood that all possible stereoisomers areencompassed by such structure. Thus, for example, the terms “compoundsof formula I,” “compounds of formula Ia,” and so forth, are intended toinclude all possible stereoisomers of the compound. Similarly, when aparticular stereoisomer is shown or named herein, it will be understoodby those skilled in the art that minor amounts of other stereoisomersmay be present in the compositions of the invention unless otherwiseindicated, provided that the utility of the composition as a whole isnot eliminated by the presence of such other isomers. Individualstereoisomers may be obtained by numerous methods that are well known inthe art, including chiral chromatography using a suitable chiralstationary phase or support, or by chemically converting them intodiastereoisomers, separating the diastereoisomers by conventional meanssuch as chromatography or recrystallization, then regenerating theoriginal stereoisomer.

Additionally, where applicable, all cis-trans or E/Z isomers (geometricisomers), tautomeric forms and topoisomeric forms of the compounds ofthe invention are included within the scope of the invention unlessotherwise specified.

More specifically, compounds of formula I can contain at least twochiral centers, which are indicated by the symbols * and ** in thefollowing formula:

In one stereoisomer, both carbon atoms identified by the * and **symbols have the (R) configuration. In this embodiment, compounds havethe (R,R) configuration at the * and ** carbon atoms or are enriched ina stereoisomeric form having the (R,R) configuration at these carbonatoms:

In another stereoisomer, both carbon atoms identified by the * and **symbols have the (S) configuration. In this embodiment, compounds havethe (S,S) configuration at the * and ** carbon atoms or are enriched ina stereoisomeric form having the (S,S) configuration at these carbonatoms:

In yet another stereoisomer, the carbon atom identified by the symbol *has the (S) configuration and the carbon atom identified by the symbol** has the (R) configuration. In this embodiment, compounds have the(S,R) configuration at the * and ** carbon atoms or are enriched in astereoisomeric form having the (S,R) configuration at these carbonatoms:

In still another stereoisomer, the carbon atom identified by thesymbol * has the (R) configuration and the carbon atom identified by thesymbol ** has the (S) configuration. In this embodiment, compounds havethe (R,S) configuration at the * and ** carbon atoms or are enriched ina stereoisomeric form having the (R,S) configuration at these carbonatoms:

In some embodiments, in order to optimize the therapeutic activity ofthe compounds of the invention, e.g., to treat hypertension, it may bedesirable that the carbon atoms identified by the * and ** symbols havea particular configuration or are enriched in a stereoisomeric formhaving such configuration. Thus, in certain aspects, this inventionrelates to each individual enantiomer or to an enantiomer-enrichedmixture of enantiomers comprising predominately one enantiomer or theother enantiomer. In other embodiments, the compounds of the inventionare present as racemic mixtures of enantiomers.

The compounds of the invention, as well as those compounds used in theirsynthesis, may also include isotopically-labeled compounds, that is,where one or more atoms have been enriched with atoms having an atomicmass different from the atomic mass predominately found in nature.Examples of isotopes that may be incorporated into the compounds offormula I, for example, include, but are not limited to, ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ³⁶Cl, and ¹⁸F. Of particular interest arecompounds of formula I enriched in tritium or carbon-14 which can beused, for example, in tissue distribution studies; compounds of formulaI enriched in deuterium especially at a site of metabolism resulting,for example, in compounds having greater metabolic stability; andcompounds of formula I enriched in a positron emitting isotope, such as¹¹C, ¹⁸F, ¹⁵O and ¹³N, which can be used, for example, in PositronEmission Topography (PET) studies.

The nomenclature used herein to name the compounds of the invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially available AutoNom software (MDL, San Leandro,Calif.).

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of theinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of the invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from the invention unless specifically indicated.

In one aspect, this invention relates to compounds of formula I:

The R¹ moiety is selected from:

H;

C₁₋₈alkyl optionally substituted with one or more fluoro atoms, e.g.,—CH₃, —CH₂CH₃, —CH₂CF₃, —(CH₂)₂CH₃, —(CH₂)₂CF₃, —CH₂CF₂CH₃, —CH₂CF₂CF₃,—CH(CH₃)₂, —C(CH₃)₃, —C(CH₃)(CF₃)₂, —CH₂CH(CH₃)₂, —(CH₂)₃CH₃,—CH(CH₂CH₃)CF₃, —CH(CH₃)CF₂CF₃, —(CH₂)₄CH₃, —(CH₂)₂CH(CH₃)₂, —(CH₂)₅CH₃,and —(CH₂)₆CH₃;

C₁₋₃ alkylene-C₆₋₁₀aryl, e.g., benzyl;

C₁₋₃ alkylene-C₁₋₉heteroaryl, e.g., —CH₂-pyridine and —(CH₂)₂-pyridine;

C₃₋₇cycloalkyl, e.g., cyclopentyl;

C₂₋₃alkylene-OH;

—[(CH₂)₂O]₁₋₃CH₃, e.g., —(CH₂)₂OCH₃ and —[(CH₂)₂O]₂CH₃;

C₁₋₆alkylene-OC(O)R²⁰, e.g., —CH₂OC(O)CH₃, —CH₂OC(O)CH₂CH₃,—CH₂OC(O)(CH₂)₂CH₃, —CH₂CH(CH₃)OC(O)CH₂CH₃, —CH₂OC(O)OCH₃,—CH₂OC(O)OCH₂CH₃, —CH(CH₃)OC(O)OCH₂CH₃, —CH(CH₃)OC(O)O—CH(CH₃)₂,—CH₂CH(CH₃)OC(O)-cyclopentyl, —CH₂OC(O)O-cyclopropyl,—CH(CH₃)—OC(O)—O-cyclohexyl, —CH₂OC(O)O-cyclopentyl,—CH₂CH(CH₃)OC(O)-phenyl, —CH₂OC(O)O-phenyl, —CH₂OC(O)—CH[CH(CH₃)₂]-NH₂,—CH₂OC(O)—CH[CH(CH₃)₂]-NHC(O)OCH₃, and —CH(CH₃)OC(O)—CH(NH₂)CH₂COOCH₃;

C₁₋₆alkylene-NR²¹R²², e.g., —(CH₂)₂—N(CH₃)₂, —(CH₂)₃—N(CH₃)₂,—(CH₂)₄—N(CH₃)₂,

—CH₂CH(NH₂)COOCH₃;

C₁₋₆alkylene-C(O)R²³, e.g., —CH₂C(O)OCH₃, —CH₂C(O)O-benzyl,—CH₂C(O)—N(CH₃)₂, and

C₀₋₆alkylenemorpholine, e.g., —(CH₂)₂-morpholine and —(CH₂)₃-morpholine:

C₁₋₆alkylene-SO₂—C₁₋₆alkyl, e.g., —(CH₂)₂SO₂CH₃;

The R²⁰ moiety is selected from:

C₁₋₆ alkyl, e.g., —CH₃ and —CH₂CH₃;

—O—C₁₋₆alkyl, e.g., —OCH₃, —O—CH₂CH₃, and —O—CH(CH₃)₂;

C₃₋₇cycloalkyl, e.g., cyclopentyl);

—O—C₃₋₇cycloalkyl, e.g., —O-cyclopropyl, —O-cyclohexyl, and—O-cyclopentyl;

phenyl;

—O-phenyl;

—NR²¹R²²;

—CH(R²⁵)—NH₂, e.g., —CH[CH(CH₃)₂]-NH₂;

—CH(R²⁵)—NHC(O)O—C₁₋₆alkyl, e.g., —CH[CH(CH₃)₂]-NHC(O)OCH₃; and

—CH(NH₂)CH₂COOCH₃.

The R²¹ and R²² moieties are independently selected from H, —C₁₋₆alkyl(e.g., —CH₃), and benzyl. Alternately, the R²¹ and R²² moieties can betaken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—, forexample to form a group such as:

The R²³ moiety is selected from —O—C₁₋₆alkyl (e.g., —OCH₃), —O-benzyl,and —NR²¹R²² (e.g., —N(CH₃)₂). The R²⁴ moiety is C₁₋₆alkyl (e.g., —CH₃and —C(CH₃)₃) or C₀₋₆alkylene-C₆₋₁₀aryl. The R²⁵ moiety is H, —CH₃,—CH(CH₃)₂, phenyl, or benzyl.

In one embodiment, R¹ is H. In other embodiments these compounds haveformulas II-VII and a-f.

In another embodiment, R¹ is selected from C₁₋₈alkyl optionallysubstituted with one or more fluoro atoms; C₁₋₃alkylene-C₆₋₁₀aryl; C₁₋₃alkylene-C₁₋₉heteroaryl; C₃₋₇ cycloalkyl; C₂₋₃alkylene-OH;—[(CH₂)₂O]₁₋₃CH₃; C₁₋₆alkylene-OC(O)R²⁰; C₁₋₆alkylene-NR²¹R²²;—CH₂CH(NH₂)—COOCH₃; C₁₋₆alkylene-C(O)R²³; C₀₋₆alkylenemorpholine;C₁₋₆alkylene-SO₂—C₁₋₆alkyl;

In other embodiments these compounds have formulas II-VII and a-f. Inone aspect of the invention, these compounds may find particular utilityas prodrugs or as intermediates in the synthetic procedures describedherein. Specific examples of such prodrug moieties include where R¹ isC₁₋₆alkylene-OC(O)R²⁰, such as —CH(CH₃)OC(O)—O-cyclohexyl:

making the compound a cilexetil ester; or R¹ is C₀₋₆alkylenemorpholinesuch as —(CH₂)₂-morpholine:

making the compound a 2-morpholinoethyl or mofetil ester; or

such as —CH₂-5-methyl-[1,3]dioxol-2-one:

making the compound a medoxomil ester.

The R² group is —OH, —CH₂OH, or —CH₂—O—C₁₋₆alkyl, and the R³ group is Hor —CH₃. Thus, the compounds of the invention may have one of thefollowing formulas:

The R⁴ and R⁵ groups are independently selected from hydrogen, halo(e.g., Cl and F), —OH, —CH₃, —OCH₃, —CN, and —CF₃. In one embodiment, R⁴is hydrogen or halo. In one embodiment, R⁵ is halo. In anotherembodiment, R⁴ is hydrogen and R⁵ is halo; or R⁴ is halo and R⁵ is halo.In other embodiments these compounds have formulas II-VII and a-f.

The X group is selected from H, —C(O)—R⁶, —C(O)—NR⁷R⁸,—C(O)—NR⁹—NR¹⁰R¹¹, —C(O)—NR¹²—NR¹³—C(O)—R¹⁴, or —CH(R¹⁵)—OR¹⁶. In oneembodiment, X is —C(O)—R⁶, which can be depicted as formula a:

In the embodiment of formula a, the R⁶ moiety is selected from C₁₋₆alkyl(e.g., —CH₃, —CH₂CH₃ and —CH₂CH(CH₃)₂), C₁₋₆alkylene-O—C₁₋₆alkyl,C₆₋₁₀aryl (e.g., phenyl), benzyl, and C₁₋₉heteroaryl (e.g., pyridine).In one embodiment, R⁶ is C₁₋₆alkyl (e.g., —CH₂CH₃ or —CH₂CH(CH₃)₂) orbenzyl. In other embodiments these compounds have formulas II-VII; andin one particular embodiment, formula II.

In one embodiment, X is —C(O)—NR⁷R⁸, which can be depicted as formula b:

In the embodiment of formula b, the R⁷ moiety is selected from H, —OH,or C₁₋₆ alkyl (e.g., —CH₃). In one embodiment, R⁷ is H, —OH, or —CH₃. Inother embodiments these compounds have formulas II-VII; and in oneparticular embodiment, formula II.

The R⁸ moiety is selected from: C₁₋₆alkyl; —O—C₁₋₆alkyl; C₅₋₆cycloalkyl;C₆₋₁₀aryl; —O—C₆₋₁₀aryl (e.g., —O-phenyl); —O-benzyl; pyridineoptionally substituted with halo, —OH, C₁₋₆alkyl, or —O—C₁₋₆alkyl;morpholine; and isoxazolidinone, for example, isoxazolidin-3-one:

In one embodiment, R⁸ is C₁₋₆alkyl (e.g., —CH₃), —O—C₁₋₆alkyl (e.g.,—O—CH₃), C₅₋₆cycloalkyl (e.g., cyclopentyl), phenyl, —O-benzyl,pyridine, pyridine substituted with halo (e.g., Br), pyridinesubstituted with C₁₋₆alkyl (e.g., —CH₃), morpholine, or isoxazolidinone.In other embodiments these compounds have formulas II-VII.

Alternately, the R⁷ and R⁸ groups can be taken together to form a ringselected from the group consisting of:

The “a” integer can be is 1 and R²⁶ is —OH. The “a” integer can be 2 andeach R²⁶ is independently halo (e.g., F) or —C₁₋₃alkylene-OH. The R²⁷moiety is —C₁₋₃alkylene-OH (e.g., —CH₂—OH), —C(O)NH₂, or —SO₂CH₃. The“b” integer is 0, or the “b” integer is 1 and R²⁸ is —C₁₋₃alkylene-OH(e.g., —CH₂—OH), or the “b” integer is 2 and each R²⁸ is C₁₋₆alkyl(e.g., —CH₃). The R²⁹ moiety is halo (e.g., fluoro), —COOH, —OH,—C₁₋₃alkylene-OH, —CH₂O—CH₃, —CONH₂, —CN, or pyridine. The R³⁰ moiety isC₁₋₆alkyl (e.g., —CH₃) or C₃₋₇cycloalkyl. The R³¹ moiety is —OH or—C₁₋₃alkylene-OH. The R³² moiety is halo, C₁₋₆alky (e.g., —CH₃);C₂₋₄alkylene-O—C₁₋₆alkyl (e.g., —(CH₂)₂OCH₂CH₃); —C(O)O—C₁₋₆alkyl (e.g.,—C(O)OCH₃ and —C(O)OCH₂CH₃); —C(O)N(CH₃)₂; pyridine; —SO₂CH₃;—C(O)-furan; or phenyl substituted with halo, —O—C₁₋₆alkyl, or —CN. TheR³³ moiety is H, —OH, —O—C₁₋₆alkyl or —O—C₆₋₁₀aryl. In other embodimentsthese compounds have formulas II-VII.

In one embodiment, R⁷ and R⁸ are taken together to form a ring selectedfrom the group consisting of:

where a is 1 and R²⁶ is —OH, or a is 2 and each R²⁶ is halo; R²⁷ is—CH₂—OH, —C(O)NH₂, or —SO₂CH₃; b is 0, or b is 1 and R²⁸ is —CH₂—OH, orb is 2 and each R²⁸ is —CH₃; R²⁹ is fluoro, —COOH, —OH, —CH₂OH,—(CH₂)₂OH, —CH₂O—CH₃, —CONH₂, —CN, or pyridine; R³⁰ is —CH₃; R³¹ is —OH;R³² is —CH₃, —(CH₂)₂OCH₂CH₃, —C(O)OCH₃, —C(O)OCH₂CH₃, —C(O)N(CH₃)₂,—SO₂CH₃, —C(O)-furan, or phenyl substituted with —CN; and R³³ is H or—OH. In other embodiments these compounds have formulas II-VII.

In one embodiment, X is —C(O)—NR⁹—NR¹⁰R¹¹, which can be depicted asformula c:

In the embodiment of formula c, the R⁹ moiety is H or C₁₋₆alkyl; and inone embodiment, R⁹ is H. The R¹⁰ moiety is H or C₁₋₆alkyl; and in oneembodiment, R¹⁰ is H. The R¹¹ moiety is selected from C₁₋₆alkyl;C₁₋₉heteroaryl optionally substituted with halo, —OH, C₁₋₆alkyl (e.g.,—CH₃), or —O—C₁₋₆alkyl (e.g., pyridine, pyridine substituted with —CH₃,pyrimidine, pyrimidine substituted with —OH, furan, thiophene, pyrazole,and pyrrole); dihydroimidazole; and phenyl optionally substituted withone or two groups selected from halo (e.g., Cl, F, Br), C₁₋₆alkyl (e.g.,—CH₃), —O—C₁₋₆alkyl (e.g., —OCH₃), and —NO₂. Exemplary optionallysubstituted pyridine groups include 2-pyridyl, 3-pyridyl, and 4-pyridyl:

as well as 3-pyridine substituted with —CH₃:

Exemplary pyrimidine groups substituted with —OH include:

An exemplary dihydroimidazole group is 4,5-dihydro-1H-imidazole:

Exemplary optionally substituted phenyl groups include: phenyl,2-chlorophenyl, 2-fluorophenyl, 3, chlorophenyl, 2-methylephenyl,3-methylphenyl, 4-methylphenyl, 4-chlorophenyl, 4-fluorophenyl,4-bromophenyl, 4-methoxyphenyl, and 2,4-dinitrophenyl. In oneembodiment, R¹¹ is selected from C₁₋₆alkyl (e.g., —CH₃ or —CH₂CH(CH₃)₂);C₁₋₉heteroaryl optionally substituted with halo, —OH, C₁₋₆alkyl, or—O—C₁₋₆alkyl (e.g., pyridine or pyrimidine substituted with —OH;dihydroimidazole; and phenyl optionally substituted with one or twogroups selected from halo (e.g., Cl, F, Br), C₁₋₆alkyl (e.g., —CH₃),—O—C₁₋₆alkyl (e.g., —OCH₃), and —NO₂. In other embodiments thesecompounds have formulas II-VII; and in one particular embodiment,formula II.

In one embodiment, X is —C(O)—NR¹²—NR¹³—C(O)—R¹⁴, which can be depictedas formula d:

In the embodiment of formula d, the R¹² moiety is H or C₁₋₆alkyl; and inone embodiment, R¹² is H. The R¹³ moiety is H or C₁₋₆alkyl; and in oneembodiment, R¹³ is H. The R¹⁴ moiety is selected from —O-benzyl;pyridine optionally substituted with halo (e.g., Cl), —OH, C₁₋₆alkyl(e.g., —CH₃), or —O—C₁₋₆alkyl; furan; and phenyl substituted with halo,—OH, —O—C₁₋₆alkyl, or —NO₂. Exemplary halo-substituted pyridine groupsinclude:

Exemplary substituted phenyl groups include: 2-hydroxyphenyl and2-nitrophenyl. In one embodiment, R¹⁴ is selected from —O-benzyl;pyridine; pyridine substituted with halo; furan; and phenyl substitutedwith —OH or —NO₂. In other embodiments these compounds have formulasII-VII; and in one particular embodiment, formula II.

In one embodiment, X is —CH(R¹⁵)—OR¹⁶, which can be depicted as formulae:

As noted above, the R¹⁵ moiety is selected from H and C₁₋₆alkyl; and inone particular embodiment of formula (e), R¹⁵ is H. The R¹⁶ moiety isselected from H, C₁₋₆alkyl, —[(CH₂)₂O]₁₋₃CH₃, C₁₋₉heteroaryl (e.g.,pyridine such as 3-pyridine), benzyl, and C₆₋₁₀aryl (e.g., phenyl)optionally substituted with —OH or —OCH₃. Alternately, the R¹⁵ and R¹⁶groups can be taken together to form —(CH₂)₄—, i.e., R¹⁵ and R¹⁶ aretaken together to form:

In one embodiment of formula (e), R¹⁶ is selected from H, —CH₃,—CH(CH₃)₂, —CH₂CH₃, pyridine, benzyl, phenyl, phenyl substituted with—OH, and phenyl substituted with —OCH₃; or R¹⁵ and R¹⁶ are takentogether to form —(CH₂)₄—. In other embodiments these compounds haveformulas II-VII; and in one particular embodiment, formula II or V.

In another embodiment, X is H, which can be depicted as formula f:

In other embodiments these compounds have formulas II-VII; and in oneparticular embodiment, formula V.

In addition, particular compounds of formula I that are of interestinclude those set forth in the Examples below, as well aspharmaceutically acceptable salts thereof.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods, the proceduresset forth in the Examples, or by using other methods, reagents, andstarting materials that are known to those of ordinary skill in the art.Although the following procedures may illustrate a particular embodimentof the invention, it is understood that other embodiments of theinvention can be similarly prepared using the same or similar methods orby using other methods, reagents and starting materials known to thoseof ordinary skill in the art. It will also be appreciated that wheretypical or preferred process conditions (for example, reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. In some instances, reactions were conducted at roomtemperature and no actual temperature measurement was taken. It isunderstood that room temperature can be taken to mean a temperaturewithin the range commonly associated with the ambient temperature in alaboratory environment, and will typically be in the range of about 18°C. to about 30° C. In other instances, reactions were conducted at roomtemperature and the temperature was actually measured and recorded.While optimum reaction conditions will typically vary depending onvarious reaction parameters such as the particular reactants, solventsand quantities used, those of ordinary skill in the art can readilydetermine suitable reaction conditions using routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions and reagents for protection anddeprotection of such functional groups are well-known in the art.Protecting groups other than those illustrated in the proceduresdescribed herein may be used, if desired. For example, numerousprotecting groups, and their introduction and removal, are described inT. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis,Fourth Edition, Wiley, New York, 2006, and references cited therein.

Carboxy protecting groups are suitable for preventing undesiredreactions at a carboxy group, and examples include, but are not limitedto, methyl, ethyl, t-butyl, benzyl (Bn), p-methoxybenzyl (PMB),9-fluorenylmethyl (Fm), trimethylsilyl (TMS), t-butyldimethylsilyl(TBDMS), diphenylmethyl (benzhydryl, DPM) and the like. Amino protectinggroups are suitable for preventing undesired reactions at an aminogroup, and examples include, but are not limited to, t-butoxycarbonyl(BOC), trityl (Tr), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl(Fmoc), formyl, trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), andthe like.

Standard deprotection techniques and reagents are used to remove theprotecting groups, and may vary depending upon which group is used. Forexample, sodium or lithium hydroxide is commonly used when thecarboxy-protecting group is methyl, an acid such as TFA or HCl iscommonly used when the carboxy-protecting group is ethyl or t-butyl, andH₂/Pd/C may be used when the carboxy-protecting group is benzyl. A BOCamino-protecting group can be removed using an acidic reagent such asTFA in DCM or HCl in 1,4-dioxane, while a Cbz amino-protecting group canbe removed by employing catalytic hydrogenation conditions such as H₂ (1atm) and 10% Pd/C in an alcoholic solvent (“H₂/Pd/C”).

Suitable bases for use in these schemes include, by way of illustrationand not limitation, potassium carbonate, calcium carbonate, sodiumcarbonate, triethylamine, pyridine, 1,8-diazabicyclo-[5.4.0]undec-7-ene(DBU), N,N-diisopropylethylamine (DIPEA), 4-methylmorpholine, sodiumhydroxide, potassium hydroxide, potassium t-butoxide, and metalhydrides.

Suitable inert diluents or solvents for use in these schemes include, byway of illustration and not limitation, tetrahydrofuran (THF),acetonitrile (MeCN), N,N-dimethylformamide (DMF), N,N-dimethylacetamide(DMA), dimethyl sulfoxide (DMSO), toluene, dichloromethane (DCM),chloroform (CHCl₃), carbon tetrachloride (CCl₄), 1,4-dioxane, methanol,ethanol, water, and the like.

Suitable carboxylic acid/amine coupling reagents includebenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), N,N,N′,N′-tetramethyl-0-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU),(2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate) (HCTU), 1,3-dicyclohexylcarbodiimide (DCC),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI),carbonyldiimidazole (CDI), 1-hydroxybenzotriazole (HOBt), and the like.Coupling reactions are conducted in an inert diluent in the presence ofa base such as DIPEA, and are performed under conventional amidebond-forming conditions.

All reactions are typically conducted at a temperature within the rangeof about −78 C to 100° C., for example at room temperature. Reactionsmay be monitored by use of thin layer chromatography (TLC), highperformance liquid chromatography (HPLC), and/or LCMS until completion.Reactions may be complete in minutes, or may take hours, typically from1-2 hours and up to 48 hours. Upon completion, the resulting mixture orreaction product may be further treated in order to obtain the desiredproduct. For example, the resulting mixture or reaction product may besubjected to one or more of the following procedures: concentrating orpartitioning (for example, between EtOAc and water or between 5% THF inEtOAc and 1M phosphoric acid); extraction (for example, with EtOAc,CHCl₃, DCM, chloroform); washing (for example, with saturated aqueousNaCl, saturated aqueous NaHCO₃, Na₂CO₃ (5%), CHCl₃ or 1M NaOH); drying(for example, over MgSO₄, over Na₂SO₄, or in vacuo); filtering;crystallizing (for example, from EtOAc and hexanes); being concentrated(for example, in vacuo); and/or purification (e.g., silica gelchromatography, flash chromatography, preparative HPLC, reversephase-HPLC, or crystallization).

Compounds of formula a, as well as their salts, can be prepared as shownin Scheme A:

The process comprises the step of coupling compound 1 with compound 2,where R², R³, R⁴, R⁵, and R⁶ are as defined for formula I, and P¹ is Hor a suitable carboxy protecting group, examples of which include, byway of illustration and not limitation, methyl, ethyl, t-butyl, benzyl,p-methoxybenzyl, 9-fluorenylmethyl, trimethylsilyl,t-butyldimethylsilyl, and diphenylmethyl. When P¹ is a carboxyprotecting group, the process further comprises removal of the P¹ groupby deprotection, in situ, with or after the coupling step. Methods ofpreparing compound 1 are described in the Examples. Compound 2 isgenerally commercially available or can be prepared using proceduresthat are known in the art.

Compounds of formula b, as well as their salts, can be prepared as shownin Scheme B:

The process comprises the step of coupling compound 3 with compound 4,where R², R³, R⁴, R⁵, R⁷, and R⁸ are as defined for formula I, and P¹ isH or a suitable carboxy protecting group. The process may furthercomprises removal of the P¹ group by deprotection, in situ, with orafter the coupling step. Methods of preparing compound 3 are describedin the Examples. Compound 4 is generally commercially available or canbe prepared using procedures that are known in the art.

Compounds of formula c, as well as their salts, can be prepared as shownin Scheme C:

The process comprises the step of coupling compound 3 with compound 5,where R², R³, R⁴, R⁵, R⁹, R¹⁰, and R¹¹ are as defined for formula I, andP¹ is H or a suitable carboxy protecting group. The process may furthercomprises removal of the P¹ group by deprotection, in situ, with orafter the coupling step. Compound 5 is generally commercially availableor can be prepared using procedures that are known in the art.

Compounds of formula d, as well as their salts, can be prepared as shownin Scheme D:

The process comprises the step of coupling compound 3 with compound 6,where R², R³, R⁴, R⁵, R¹², R¹³, and R¹⁴ are as defined for formula I,and P¹ is H or a suitable carboxy protecting group. The process mayfurther comprises removal of the P¹ group by deprotection, in situ, withor after the coupling step. Compound 6 is generally commerciallyavailable or can be prepared using procedures that are known in the art.

Compounds of formula e, as well as their salts, can be prepared as shownin Scheme E:

The process comprises the step of coupling compound 1 with compound 7 orcompound 8, where R², R³, R⁴, R⁵, R¹⁵, and R¹⁶ are as defined forformula I, and P¹ is H or a suitable carboxy protecting group. Theprocess may further comprises removal of the P¹ group by deprotection,in situ, with or after the coupling step. Compound 8 is generallycommercially available or can be prepared using procedures that areknown in the art.

Compounds of formula f, as well as their salts, can be prepared as shownin Scheme F:

The process comprises the step of coupling compound 1 with formic acid,where R², R³, R⁴, and R⁵, are as defined for formula I, and P¹ is H or asuitable carboxy protecting group. The process may further comprisesremoval of the P¹ group by deprotection, in situ, with or after thecoupling step.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereof are described in the Examples set forth below.

Utility

Compounds of the invention possess neprilysin (NEP) inhibition activity,that is, the compounds are able to inhibit enzyme-catalytic activity. Inanother embodiment, the compounds do not exhibit significant inhibitoryactivity of the angiotensin-converting enzyme. One measure of theability of a compound to inhibit NEP activity is the inhibition constant(pK_(i)). The pK_(i) value is the negative logarithm to base 10 of thedissociation constant (K_(i)), which is typically reported in molarunits. Compounds of the invention of particular interest are thosehaving a pK_(i) at NEP greater than or equal to 6.0, particularly thosehaving a pK_(i) greater than or equal to 7.0, and even more particularlythose having a pK_(i) greater than or equal to 8.0. In one embodiment,compounds of interest have a pK_(i) in the range of 6.0-6.9; in anotherembodiment, compounds of interest have a pK_(i) in the range of 7.0-7.9;in yet another embodiment, compounds of interest have a pK_(i) in therange of 8.0-8.9; and in still another embodiment, compounds of interesthave a pK_(i) in the range of greater than or equal to 9.0. Such valuescan be determined by techniques that are well known in the art, as wellas in the assays described herein.

Another measure of the ability of a compound to inhibit NEP activity isthe apparent inhibition constant (IC₅₀), which is the molarconcentration of compound that results in half-maximal inhibition ofsubstrate conversion by the NEP enzyme. The pIC₅₀ value is the negativelogarithm to base 10 of the IC₅₀. Compounds of the invention that are ofparticular interest, include those that exhibit a pIC₅₀ for NEP greaterthan or equal to about 5.0. Compounds of interest also include thosehaving a pIC₅₀ for NEP≧about 6.0 or a pIC₅₀ for NEP≧about 7.0. Inanother embodiment, compounds of interest have a pIC₅₀ for NEP withinthe range of about 7.0-11.0; and in another embodiment, within the rangeof about 8.0-11.0, such as within the range of about 8.0-10.0.

It is noted that in some cases, compounds of the invention may possessweak NEP inhibition activity. In such cases, those of skill in the artwill recognize that these compounds still have utility as researchtools.

Exemplary assays to determine properties of compounds of the invention,such as the NEP inhibiting activity, are described in the Examples andinclude by way of illustration and not limitation, assays that measureNEP inhibition (described in Assay 1).

Useful secondary assays include assays to measure ACE inhibition (alsodescribed in Assay 1) and aminopeptidase P (APP) inhibition (describedin Sulpizio et al. (2005) JPET 315:1306-1313). A pharmacodynamic assayto assess the in vivo inhibitory potencies for ACE and NEP inanesthetized rats is described in Assay 2 (see also Seymour et al.(1985) Hypertension 7(Suppl I):I-35-I-42 and Wigle et al. (1992) Can. J.Physiol. Pharmacol. 70:1525-1528), where ACE inhibition is measured asthe percent inhibition of the angiotensin I pressor response and NEPinhibition is measured as increased urinary cyclic guanosine 3′,5′-monophosphate (cGMP) output.

There are many in vivo assays that can be used to ascertain furtherutilities of the compounds of the invention. The conscious spontaneouslyhypertensive rat (SHR) model is a renin dependent hypertension model,and is described in Assay 3. See also Intengan et al. (1999) Circulation100(22):2267-2275 and Badyal et al. (2003) Indian Journal ofPharmacology 35:349-362. The conscious desoxycorticosterone acetate-salt(DOCA-salt) rat model is a volume dependent hypertension model that isuseful for measuring NEP activity, and is described in Assay 4. See alsoTrapani et al. (1989) J. Cardiovasc. Pharmacol. 14:419-424, Intengan etal. (1999) Hypertension 34(4):907-913, and Badyal et al. (2003) supra).The DOCA-salt model is particularly useful for evaluating the ability ofa test compound to reduce blood pressure as well as to measure a testcompound's ability to prevent or delay a rise in blood pressure. TheDahl salt-sensive (DSS) hypertensive rat model is a model ofhypertension that is sensitive to dietary salt (NaCl), and is describedin Assay 5. See also Rapp (1982) Hypertension 4:753-763. The ratmonocrotaline model of pulmonary arterial hypertension described, forexample, in Kato et al. (2008) J. Cardiovasc. Pharmacol. 51(1):18-23, isa reliable predictor of clinical efficacy for the treatment of pulmonaryarterial hypertension. Heart failure animal models include the DSS ratmodel for heart failure and the aorto-caval fistula model (AV shunt),the latter of which is described, for example, in Norling et al. (1996)J. Amer. Soc. Nephrol. 7:1038-1044. Other animal models, such as the hotplate, tail-flick and formalin tests, can be used to measure theanalgesic properties of compounds of the invention, as well as thespinal nerve ligation (SNL) model of neuropathic pain. See, for example,Malmberg et al. (1999) Current Protocols in Neuroscience 8.9.1-8.9.15.

Compounds of the invention are expected to inhibit the NEP enzyme in anyof the assays listed above, or assays of a similar nature. Thus, theaforementioned assays are useful in determining the therapeutic utilityof compounds of the invention, for example, their utility asantihypertensive agents or antidiarrheal agents. Other properties andutilities of compounds of the invention can be demonstrated using otherin vitro and in vivo assays well-known to those skilled in the art.Compounds of formula I may be active drugs as well as prodrugs. Thus,when discussing the activity of compounds of the invention, it isunderstood that any such prodrugs may not exhibit the expected activityin an assay, but are expected to exhibit the desired activity oncemetabolized.

Compounds of the invention are expected to be useful for the treatmentand/or prevention of medical conditions responsive to NEP inhibition.Thus it is expected that patients suffering from a disease or disorderthat is treated by inhibiting the NEP enzyme or by increasing the levelsof its peptide substrates, can be treated by administering atherapeutically effective amount of a compound of the invention. Forexample, by inhibiting NEP, the compounds are expected to potentiate thebiological effects of endogenous peptides that are metabolized by NEP,such as the natriuretic peptides, bombesin, bradykinins, calcitonin,endothelins, enkephalins, neurotensin, substance P and vasoactiveintestinal peptide. Thus, these compounds are expected to have otherphysiological actions, for example, on the renal, central nervous,reproductive and gastrointestinal systems.

In one embodiment of the invention, patients suffering from a disease ordisorder that is treated by inhibiting the NEP enzyme, are treated byadministering a compound of the invention that is in its active form,i.e., a compound of formula I where R¹ is H, and R², R³, R⁴, R⁵, R⁶, andX are as defined for formula I.

In another embodiment, patients are treated by administering a compoundthat is metabolized in vitro to form a compound of formula I where R¹ isH, and R², R³, R⁴, R⁵, R⁶, and X are as defined for formula I.

In another embodiment, patients are treated by administering a compoundof the invention that is in its prodrug form at the R¹ group, i.e., acompound of formula I where R¹ is selected from C₁₋₈alkyl optionallysubstituted with one or more fluoro atoms; C₁₋₃alkylene-C₆₋₁₀aryl;C₁₋₃alkylene-C₁₋₉heteroaryl; C₃₋₇cycloalkyl; C₂₋₃alkylene-OH;—[(CH₂)₂O]₁₋₃CH₃; C₁₋₆alkylene-OC(O)R²⁰; C₁₋₆alkylene-NR²¹R²²;—CH₂CH(NH₂)—COOCH₃; C₁₋₆alkylene-C(O)R²³; C₀₋₆alkylenemorpholine;C₁₋₆alkylene-SO₂—C₁₋₆alkyl;

Cardiovascular Diseases

By potentiating the effects of vasoactive peptides like the natriureticpeptides and bradykinin, compounds of the invention are expected to findutility in treating and/or preventing medical conditions such ascardiovascular diseases. See, for example, Rogues et al. (1993)Pharmacol. Rev. 45:87-146 and Dempsey et al. (2009) Amer. J. ofPathology 174(3):782-796. Cardiovascular diseases of particular interestinclude hypertension and heart failure. Hypertension includes, by way ofillustration and not limitation: primary hypertension, which is alsoreferred to as essential hypertension or idiopathic hypertension;secondary hypertension; hypertension with accompanying renal disease;severe hypertension with or without accompanying renal disease;pulmonary hypertension, including pulmonary arterial hypertension; andresistant hypertension. Heart failure includes, by way of illustrationand not limitation: congestive heart failure; acute heart failure;chronic heart failure, for example with reduced left ventricularejection fraction (also referred to as systolic heart failure) or withpreserved left ventricular ejection fraction (also referred to asdiastolic heart failure); and acute and chronic decompensated heartfailure, with or without accompanying renal disease. Thus, oneembodiment of the invention relates to a method for treatinghypertension, particularly primary hypertension or pulmonary arterialhypertension, comprising administering to a patient a therapeuticallyeffective amount of a compound of the invention.

For treatment of primary hypertension, the therapeutically effectiveamount is typically the amount that is sufficient to lower the patient'sblood pressure. This would include both mild-to-moderate hypertensionand severe hypertension. When used to treat hypertension, the compoundmay be administered in combination with other therapeutic agents such asaldosterone antagonists, aldosterone synthase inhibitors,angiotensin-converting enzyme inhibitors and dual-actingangiotensin-converting enzyme/neprilysin inhibitors,angiotensin-converting enzyme 2 (ACE2) activators and stimulators,angiotensin-II vaccines, anti-diabetic agents, anti-lipid agents,anti-thrombotic agents, AT₁ receptor antagonists and dual-acting AT₁receptor antagonist/neprilysin inhibitors, β₁-adrenergic receptorantagonists, dual-acting β-adrenergic receptor antagonist/α₁-receptorantagonists, calcium channel blockers, diuretics, endothelin receptorantagonists, endothelin converting enzyme inhibitors, neprilysininhibitors, natriuretic peptides and their analogs, natriuretic peptideclearance receptor antagonists, nitric oxide donors, non-steroidalanti-inflammatory agents, phosphodiesterase inhibitors (specificallyPDE-V inhibitors), prostaglandin receptor agonists, renin inhibitors,soluble guanylate cyclase stimulators and activators, and combinationsthereof. In one particular embodiment of the invention, a compound ofthe invention is combined with an AT₁ receptor antagonist, a calciumchannel blocker, a diuretic, or a combination thereof, and used to treatprimary hypertension. In another particular embodiment of the invention,a compound of the invention is combined with an AT₁ receptor antagonist,and used to treat hypertension with accompanying renal disease. Whenused to treat resistant hypertension, the compound may be administeredin combination with other therapeutic agents such as aldosteronesynthase inhibitors.

For treatment of pulmonary arterial hypertension, the therapeuticallyeffective amount is typically the amount that is sufficient to lower thepulmonary vascular resistance. Other goals of therapy are to improve apatient's exercise capacity. For example, in a clinical setting, thetherapeutically effective amount can be the amount that improves apatient's ability to walk comfortably for a period of 6 minutes(covering a distance of approximately 20-40 meters). When used to treatpulmonary arterial hypertension the compound may be administered incombination with other therapeutic agents such as α-adrenergic receptorantagonists, β₁-adrenergic receptor antagonists, β₂-adrenergic receptoragonists, angiotensin-converting enzyme inhibitors, anticoagulants,calcium channel blockers, diuretics, endothelin receptor antagonists,PDE-V inhibitors, prostaglandin analogs, selective serotonin reuptakeinhibitors, and combinations thereof. In one particular embodiment ofthe invention, a compound of the invention is combined with a PDE-Vinhibitor or a selective serotonin reuptake inhibitor and used to treatpulmonary arterial hypertension.

Another embodiment of the invention relates to a method for treatingheart failure, in particular congestive heart failure (including bothsystolic and diastolic congestive heart failure), comprisingadministering to a patient a therapeutically effective amount of acompound of the invention. Typically, the therapeutically effectiveamount is the amount that is sufficient to lower blood pressure and/orimprove renal functions. In a clinical setting, the therapeuticallyeffective amount can be the amount that is sufficient to improve cardiachemodynamics, like for instance reduction in wedge pressure, rightatrial pressure, filling pressure, and vascular resistance. In oneembodiment, the compound is administered as an intravenous dosage form.When used to treat heart failure, the compound may be administered incombination with other therapeutic agents such as adenosine receptorantagonists, advanced glycation end product breakers, aldosteroneantagonists, AT₁ receptor antagonists, β₁-adrenergic receptorantagonists, dual-acting (3-adrenergic receptor antagonist/α₁-receptorantagonists, chymase inhibitors, digoxin, diuretics, endothelinconverting enzyme (ECE) inhibitors, endothelin receptor antagonists,natriuretic peptides and their analogs, natriuretic peptide clearancereceptor antagonists, nitric oxide donors, prostaglandin analogs, PDE-Vinhibitors, soluble guanylate cyclase activators and stimulators, andvasopressin receptor antagonists. In one particular embodiment of theinvention, a compound of the invention is combined with an aldosteroneantagonist, a β₁-adrenergic receptor antagonist, an AT₁ receptorantagonist, or a diuretic, and used to treat congestive heart failure.

Diarrhea

As NEP inhibitors, compounds of the invention are expected to inhibitthe degradation of endogenous enkephalins and thus such compounds mayalso find utility for the treatment of diarrhea, including infectiousand secretory/watery diarrhea. See, for example, Baumer et al. (1992)Gut 33:753-758; Farthing (2006) Digestive Diseases 24:47-58; andMarçais-Collado (1987) Eur. J. Pharmacol. 144(2):125-132. When used totreat diarrhea, compounds of the invention may be combined with one ormore additional antidiarrheal agents.

Renal Diseases

By potentiating the effects of vasoactive peptides like the natriureticpeptides and bradykinin, compounds of the invention are expected toenhance renal function (see Chen et al. (1999) Circulation100:2443-2448; Lipkin et al. (1997) Kidney Int. 52:792-801; and Dussauleet al. (1993) Clin. Sci. 84:31-39) and find utility in treating and/orpreventing renal diseases. Renal diseases of particular interest includediabetic nephropathy, chronic kidney disease, proteinuria, andparticularly acute kidney injury or acute renal failure (see Sharkovskaet al. (2011) Clin. Lab. 57:507-515 and Newaz et al. (2010) RenalFailure 32:384-390). When used to treat renal disease, the compound maybe administered in combination with other therapeutic agents such asangiotensin-converting enzyme inhibitors, AT₁ receptor antagonists, anddiuretics.

Preventative Therapy

By potentiating the effects of the natriuretic peptides, compounds ofthe invention are also expected to be useful in preventative therapy,due to the antihypertrophic and antifibrotic effects of the natriureticpeptides (see Potter et al. (2009) Handbook of Experimental Pharmacology191:341-366), for example in preventing the progression of cardiacinsufficiency after myocardial infarction, preventing arterialrestenosis after angioplasty, preventing thickening of blood vesselwalls after vascular operations, preventing atherosclerosis, andpreventing diabetic angiopathy.

Glaucoma

By potentiating the effects of the natriuretic peptides, compounds ofthe invention are expected to be useful to treat glaucoma. See, forexample, Diestelhorst et al. (1989) International Ophthalmology12:99-101. When used to treat glaucoma, compounds of the invention maybe combined with one or more additional antiglaucoma agents.

Pain Relief

As NEP inhibitors, compounds of the invention are expected to inhibitthe degradation of endogenous enkephalins and thus such compounds mayalso find utility as analgesics. See, for example, Rogues et al. (1980)Nature 288:286-288 and Thanawala et al. (2008) Current Drug Targets9:887-894. When used to treat pain, the compounds of the invention maybe combined with one or more additional antinociceptive drugs such asaminopeptidase N or dipeptidyl peptidase III inhibitors, non-steroidalanti-inflammatory agents, monoamine reuptake inhibitors, musclerelaxants, NMDA receptor antagonists, opioid receptor agonists,5-HT_(1D) serotonin receptor agonists, and tricyclic antidepressants.

Other Utilities

Due to their NEP inhibition properties, compounds of the invention arealso expected to be useful as antitussive agents, as well as findutility in the treatment of portal hypertension associated with livercirrhosis (see Sansoe et al. (2005) J. Hepatol. 43:791-798), cancer (seeVesely (2005) J. Investigative Med. 53:360-365), depression (see Nobleet al. (2007) Exp. Opin. Ther. Targets 11:145-159), menstrual disorders,preterm labor, pre-eclampsia, endometriosis, reproductive disorders (forexample, male and female infertility, polycystic ovarian syndrome,implantation failure), and male and female sexual dysfunction, includingmale erectile dysfunction and female sexual arousal disorder. Morespecifically, the compounds of the invention are expected to be usefulin treating female sexual dysfunction (see Pryde et al. (2006) J. Med.Chem. 49:4409-4424), which is often defined as a female patient'sdifficulty or inability to find satisfaction in sexual expression. Thiscovers a variety of diverse female sexual disorders including, by way ofillustration and not limitation, hypoactive sexual desire disorder,sexual arousal disorder, orgasmic disorder and sexual pain disorder.When used to treat such disorders, especially female sexual dysfunction,compounds of the invention may be combined with one or more of thefollowing secondary agents: PDE-V inhibitors, dopamine agonists,estrogen receptor agonists and/or antagonists, androgens, and estrogens.Due to their NEP inhibition properties, compounds of the invention arealso expected to have anti-inflammatory properties, and are expected tohave utility as such, particularly when used in combination withstatins.

Recent studies suggest that NEP plays a role in regulating nervefunction in insulin-deficient diabetes and diet induced obesity. Coppeyet al. (2011) Neuropharmacology 60:259-266. Therefore, due to their NEPinhibition properties, compounds of the invention are also expected tobe useful in providing protection from nerve impairment caused bydiabetes or diet induced obesity.

The amount of the compound of the invention administered per dose or thetotal amount administered per day may be predetermined or it may bedetermined on an individual patient basis by taking into considerationnumerous factors, including the nature and severity of the patient'scondition, the condition being treated, the age, weight, and generalhealth of the patient, the tolerance of the patient to the active agent,the route of administration, pharmacological considerations such as theactivity, efficacy, pharmacokinetics and toxicology profiles of thecompound and any secondary agents being administered, and the like.Treatment of a patient suffering from a disease or medical condition(such as hypertension) can begin with a predetermined dosage or a dosagedetermined by the treating physician, and will continue for a period oftime necessary to prevent, ameliorate, suppress, or alleviate thesymptoms of the disease or medical condition. Patients undergoing suchtreatment will typically be monitored on a routine basis to determinethe effectiveness of therapy. For example, in treating hypertension,blood pressure measurements may be used to determine the effectivenessof treatment. Similar indicators for other diseases and conditionsdescribed herein, are well known and are readily available to thetreating physician. Continuous monitoring by the physician will insurethat the optimal amount of the compound of the invention will beadministered at any given time, as well as facilitating thedetermination of the duration of treatment. This is of particular valuewhen secondary agents are also being administered, as their selection,dosage, and duration of therapy may also require adjustment. In thisway, the treatment regimen and dosing schedule can be adjusted over thecourse of therapy so that the lowest amount of active agent thatexhibits the desired effectiveness is administered and, further, thatadministration is continued only so long as is necessary to successfullytreat the disease or medical condition.

Research Tools

Since compounds of the invention possess NEP enzyme inhibition activity,such compounds are also useful as research tools for investigating orstudying biological systems or samples having a NEP enzyme, for exampleto study diseases where the NEP enzyme or its peptide substrates plays arole. Any suitable biological system or sample having a NEP enzyme maybe employed in such studies which may be conducted either in vitro or invivo. Representative biological systems or samples suitable for suchstudies include, but are not limited to, cells, cellular extracts,plasma membranes, tissue samples, isolated organs, mammals (such asmice, rats, guinea pigs, rabbits, dogs, pigs, humans, and so forth), andthe like, with mammals being of particular interest. In one particularembodiment of the invention, NEP enzyme activity in a mammal isinhibited by administering a NEP-inhibiting amount of a compound of theinvention. Compounds of the invention can also be used as research toolsby conducting biological assays using such compounds.

When used as a research tool, a biological system or sample comprising aNEP enzyme is typically contacted with a NEP enzyme-inhibiting amount ofa compound of the invention. After the biological system or sample isexposed to the compound, the effects of inhibiting the NEP enzyme aredetermined using conventional procedures and equipment, such as bymeasuring receptor binding in a binding assay or measuringligand-mediated changes in a functional assay. Exposure encompassescontacting cells or tissue with the compound, administering the compoundto a mammal, for example by i.p., p.o, i.v. , s.c., or inhaledadministration, and so forth. This determining step can involvemeasuring a response (a quantitative analysis) or can involve making anobservation (a qualitative analysis). Measuring a response involves, forexample, determining the effects of the compound on the biologicalsystem or sample using conventional procedures and equipment, such asenzyme activity assays and measuring enzyme substrate or productmediated changes in functional assays. The assay results can be used todetermine the activity level as well as the amount of compound necessaryto achieve the desired result, that is, a NEP enzyme-inhibiting amount.Typically, the determining step will involve determining the effects ofinhibiting the NEP enzyme.

Additionally, compounds of the invention can be used as research toolsfor evaluating other chemical compounds, and thus are also useful inscreening assays to discover, for example, new compounds havingNEP-inhibiting activity. In this manner, a compound of the invention isused as a standard in an assay to allow comparison of the resultsobtained with a test compound and with compounds of the invention toidentify those test compounds that have about equal or superioractivity, if any. For example, pK_(i) data for a test compound or agroup of test compounds is compared to the pK_(i) data for a compound ofthe invention to identify those test compounds that have the desiredproperties, for example, test compounds having a pK_(i) value aboutequal or superior to a compound of the invention, if any. This aspect ofthe invention includes, as separate embodiments, both the generation ofcomparison data (using the appropriate assays) and the analysis of testdata to identify test compounds of interest. Thus, a test compound canbe evaluated in a biological assay, by a method comprising the steps of:(a) conducting a biological assay with a test compound to provide afirst assay value; (b) conducting the biological assay with a compoundof the invention to provide a second assay value; wherein step (a) isconducted either before, after or concurrently with step (b); and (c)comparing the first assay value from step (a) with the second assayvalue from step (b). Exemplary biological assays include a NEP enzymeinhibition assay.

Pharmaceutical Compositions and Formulations

Compounds of the invention are typically administered to a patient inthe form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to, oral,rectal, vaginal, nasal, inhaled, topical (including transdermal),ocular, and parenteral modes of administration. Further, the compoundsof the invention may be administered, for example orally, in multipledoses per day (for example, two, three, or four times daily), in asingle daily dose or a single weekly dose. It will be understood thatany form of the compounds of the invention, (that is, free base, freeacid, pharmaceutically acceptable salt, solvate, etc.) that is suitablefor the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of the invention. The compositions may containother therapeutic and/or formulating agents if desired. When discussingcompositions, the “compound of the invention” may also be referred toherein as the “active agent,” to distinguish it from other components ofthe formulation, such as the carrier. Thus, it is understood that theterm “active agent” includes compounds of formula I as well aspharmaceutically acceptable salts, solvates and prodrugs of thatcompound.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the invention. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,such as in bulk compositions, or less than a therapeutically effectiveamount, that is, individual unit doses designed for multipleadministration to achieve a therapeutically effective amount. Typically,the composition will contain from about 0.01-95 wt % of active agent,including, from about 0.01-30 wt %, such as from about 0.01-10 wt %,with the actual amount depending upon the formulation itself, the routeof administration, the frequency of dosing, and so forth. In oneembodiment, a composition suitable for an oral dosage form, for example,may contain about 5-70 wt %, or from about 10-60 wt % of active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers and the like usingconventional procedures and equipment.

In one embodiment, the pharmaceutical compositions are suitable for oraladministration. Suitable compositions for oral administration may be inthe form of capsules, tablets, pills, lozenges, cachets, dragees,powders, granules; solutions or suspensions in an aqueous or non-aqueousliquid; oil-in-water or water-in-oil liquid emulsions; elixirs orsyrups; and the like; each containing a predetermined amount of theactive agent.

When intended for oral administration in a solid dosage form (capsules,tablets, pills and the like), the composition will typically comprisethe active agent and one or more pharmaceutically acceptable carriers,such as sodium citrate or dicalcium phosphate. Solid dosage forms mayalso comprise: fillers or extenders, such as starches, microcrystallinecellulose, lactose, sucrose, glucose, mannitol, and/or silicic acid;binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; humectants, such as glycerol;disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as cetyl alcohol and/or glycerol monostearate; absorbents, such askaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodiumsulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, optionally with one ormore of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (for example, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.Suspensions may contain suspending agents such as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminium metahydroxide, bentonite,agar-agar and tragacanth, and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, that is, each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

In another embodiment, the compositions of the invention are suitablefor inhaled administration, and will typically be in the form of anaerosol or a powder. Such compositions are generally administered usingwell-known delivery devices, such as a nebulizer, dry powder, ormetered-dose inhaler. Nebulizer devices produce a stream of highvelocity air that causes the composition to spray as a mist that iscarried into a patient's respiratory tract. An exemplary nebulizerformulation comprises the active agent dissolved in a carrier to form asolution, or micronized and combined with a carrier to form a suspensionof micronized particles of respirable size. Dry powder inhalersadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. An exemplary dry powderformulation comprises the active agent dry-blended with an excipientsuch as lactose, starch, mannitol, dextrose, polylactic acid,polylactide-co-glycolide, and combinations thereof. Metered-doseinhalers discharge a measured amount of the active agent usingcompressed propellant gas. An exemplary metered-dose formulationcomprises a solution or suspension of the active agent in a liquefiedpropellant, such as a chlorofluorocarbon or hydrofluoroalkane. Optionalcomponents of such formulations include co-solvents, such as ethanol orpentane, and surfactants, such as sorbitan trioleate, oleic acid,lecithin, glycerin, and sodium lauryl sulfate. Such compositions aretypically prepared by adding chilled or pressurized hydrofluoroalkane toa suitable container containing the active agent, ethanol (if present)and the surfactant (if present). To prepare a suspension, the activeagent is micronized and then combined with the propellant.Alternatively, a suspension formulation can be prepared by spray dryinga coating of surfactant on micronized particles of the active agent. Theformulation is then loaded into an aerosol canister, which forms aportion of the inhaler.

Compounds of the invention can also be administered parenterally (forexample, by subcutaneous, intravenous, intramuscular, or intraperitonealinjection). For such administration, the active agent is provided in asterile solution, suspension, or emulsion. Exemplary solvents forpreparing such formulations include water, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, oils, gelatin,fatty acid esters such as ethyl oleate, and the like. Parenteralformulations may also contain one or more anti-oxidants, solubilizers,stabilizers, preservatives, wetting agents, emulsifiers, and dispersingagents. Surfactants, additional stabilizing agents or pH-adjustingagents (acids, bases or buffers) and anti-oxidants are particularlyuseful to provide stability to the formulation, for example, to minimizeor avoid hydrolysis of ester and amide linkages that may be present inthe compound. These formulations may be rendered sterile by use of asterile injectable medium, a sterilizing agent, filtration, irradiation,or heat. In one particular embodiment, the parenteral formulationcomprises an aqueous cyclodextrin solution as the pharmaceuticallyacceptable carrier. Suitable cyclodextrins include cyclic moleculescontaining six or more α-D-glucopyranose units linked at the 1,4positions by a linkages as in amylase, β-cyclodextrin orcycloheptaamylose. Exemplary cyclodextrins include cyclodextrinderivatives such as hydroxypropyl and sulfobutyl ether cyclodextrinssuch as hydroxypropyl-β-cyclodextrin and sulfobutyl ether3-cyclodextrin. Exemplary buffers for such formulations includecarboxylic acid-based buffers such as citrate, lactate and maleatebuffer solutions.

Compounds of the invention can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, thecompound can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

Secondary Agents

The compounds of the invention may be useful as the sole treatment of adisease or may be combined with one or more additional therapeuticagents in order to obtain the desired therapeutic effect. Thus, in oneembodiment, pharmaceutical compositions of the invention contain otherdrugs that are co-administered with a compound of the invention. Forexample, the composition may further comprise one or more drugs (alsoreferred to as “secondary agents(s)”). Such therapeutic agents are wellknown in the art, and include adenosine receptor antagonists,α-adrenergic receptor antagonists, β₁-adrenergic receptor antagonists,β₂-adrenergic receptor agonists, dual-acting β-adrenergic receptorantagonist/α₁-receptor antagonists, advanced glycation end productbreakers, aldosterone antagonists, aldosterone synthase inhibitors,aminopeptidase N inhibitors, androgens, angiotensin-converting enzymeinhibitors and dual-acting angiotensin-converting enzyme/neprilysininhibitors, angiotensin-converting enzyme 2 activators and stimulators,angiotensin-II vaccines, anticoagulants, anti-diabetic agents,antidiarrheal agents, anti-glaucoma agents, anti-lipid agents,antinociceptive agents, anti-thrombotic agents, AT₁ receptor antagonistsand dual-acting AT₁ receptor antagonist/neprilysin inhibitors andmultifunctional angiotensin receptor blockers, bradykinin receptorantagonists, calcium channel blockers, chymase inhibitors, digoxin,diuretics, dopamine agonists, endothelin converting enzyme inhibitors,endothelin receptor antagonists, HMG-CoA reductase inhibitors,estrogens, estrogen receptor agonists and/or antagonists, monoaminereuptake inhibitors, muscle relaxants, natriuretic peptides and theiranalogs, natriuretic peptide clearance receptor antagonists, neprilysininhibitors, nitric oxide donors, non-steroidal anti-inflammatory agents,N-methyl d-aspartate receptor antagonists, opioid receptor agonists,phosphodiesterase inhibitors, prostaglandin analogs, prostaglandinreceptor agonists, renin inhibitors, selective serotonin reuptakeinhibitors, sodium channel blocker, soluble guanylate cyclasestimulators and activators, tricyclic antidepressants, vasopressinreceptor antagonists, and combinations thereof. Specific examples ofthese agents are detailed herein.

Accordingly, in yet another aspect of the invention, a pharmaceuticalcomposition comprises a compound of the invention, a second activeagent, and a pharmaceutically acceptable carrier. Third, fourth etc.active agents may also be included in the composition. In combinationtherapy, the amount of compound of the invention that is administered,as well as the amount of secondary agents, may be less than the amounttypically administered in monotherapy.

Compounds of the invention may be physically mixed with the secondactive agent to form a composition containing both agents; or each agentmay be present in separate and distinct compositions which areadministered to the patient simultaneously or at separate times. Forexample, a compound of the invention can be combined with a secondactive agent using conventional procedures and equipment to form acombination of active agents comprising a compound of the invention anda second active agent. Additionally, the active agents may be combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition comprising a compound of the invention, a second activeagent and a pharmaceutically acceptable carrier. In this embodiment, thecomponents of the composition are typically mixed or blended to create aphysical mixture. The physical mixture is then administered in atherapeutically effective amount using any of the routes describedherein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention, ranging anywhere from concurrent withadministration of the compound of the invention to about 24 hourspost-dose. This is also referred to as sequential administration. Thus,a compound of the invention can be orally administered simultaneously orsequentially with another active agent using two tablets, with onetablet for each active agent, where sequential may mean beingadministered immediately after administration of the compound of theinvention or at some predetermined time later (for example, one hourlater or three hours later). It is also contemplated that the secondaryagent may be administered more than 24 hours after administration of thecompound of the invention. Alternatively, the combination may beadministered by different routes of administration, that is, one orallyand the other by inhalation.

In one embodiment, the kit comprises a first dosage form comprising acompound of the invention and at least one additional dosage formcomprising one or more of the secondary agents set forth herein, inquantities sufficient to carry out the methods of the invention. Thefirst dosage form and the second (or third, etc.) dosage form togethercomprise a therapeutically effective amount of active agents for thetreatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount such that they are typically administered in an amountthat produces a therapeutically beneficial effect when co-administeredwith a compound of the invention. The secondary agent can be in the formof a pharmaceutically acceptable salt, solvate, optically purestereoisomer, and so forth. The secondary agent may also be in the formof a prodrug, for example, a compound having a carboxylic acid groupthat has been esterified. Thus, secondary agents listed herein areintended to include all such forms, and are commercially available orcan be prepared using conventional procedures and reagents.

In one embodiment, compounds of the invention are administered incombination with an adenosine receptor antagonist, representativeexamples of which include, but are not limited to, naxifylline,rolofylline, SLV-320, theophylline, and tonapofylline.

In one embodiment, compounds of the invention are administered incombination with an α-adrenergic receptor antagonist, representativeexamples of which include, but are not limited to, doxazosin, prazosin,tamsulosin, and terazosin.

Compounds of the invention may also be administered in combination witha β₁-adrenergic receptor antagonist (“β₁-blockers”). Representativeβ₁-blockers include, but are not limited to, acebutolol, alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucindolol, bucumolol, bufetolol, bufuralol,bunitrolol, bupranolol, bubridine, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol, indenolol, labetolol, levobunolol, mepindolol, metipranolol,metoprolol such as metoprolol succinate and metoprolol tartrate,moprolol, nadolol, nadoxolol, nebivalol, nipradilol, oxprenolol,penbutolol, perbutolol, pindolol, practolol, pronethalol, propranolol,sotalol, sufinalol, talindol, tertatolol, tilisolol, timolol,toliprolol, xibenolol, and combinations thereof. In one particularembodiment, the β₁-antagonist is selected from atenolol, bisoprolol,metoprolol, propranolol, sotalol, and combinations thereof. Typically,the β₁-blocker will be administered in an amount sufficient to providefrom about 2-900 mg per dose.

In one embodiment, compounds of the invention are administered incombination with a β₂-adrenergic receptor agonist, representativeexamples of which include, but are not limited to, albuterol,bitolterol, fenoterol, formoterol, indacaterol, isoetharine,levalbuterol, metaproterenol, pirbuterol, salbutamol, salmefamol,salmeterol, terbutaline, vilanterol, and the like Typically, theβ₂-adrenergic receptor agonist will be administered in an amountsufficient to provide from about 0.05-500 μg per dose.

In one embodiment, compounds of the invention are administered incombination with an advanced glycation end product (AGE) breaker,examples of which include, by way of illustration and not limitation,alagebrium (or ALT-711), and TRC4149.

In another embodiment, compounds of the invention are administered incombination with an aldosterone antagonist, representative examples ofwhich include, but are not limited to, eplerenone, spironolactone, andcombinations thereof. Typically, the aldosterone antagonist will beadministered in an amount sufficient to provide from about 5-300 mg perday.

In one embodiment, compounds of the invention are administered incombination with an aminopeptidase N or dipeptidyl peptidase IIIinhibitor, examples of which include, by way of illustration and notlimitation, bestatin and PC18 (2-amino-4-methylsulfonyl butane thiol,methionine thiol).

Compounds of the invention can also be administered in combination withan angiotensin-converting enzyme (ACE) inhibitor. Representative ACEinhibitors include, but are not limited to, accupril, alacepril,benazepril, benazeprilat, captopril, ceranapril, cilazapril, delapril,enalapril, enalaprilat, fosinopril, fosinoprilat, imidapril, lisinopril,moexipril, monopril, moveltipril, pentopril, perindopril, quinapril,quinaprilat, ramipril, ramiprilat, saralasin acetate, spirapril,temocapril, trandolapril, zofenopril, and combinations thereof. In aparticular embodiment, the ACE inhibitor is selected from: benazepril,captopril, enalapril, lisinopril, ramipril, and combinations thereof.Typically, the ACE inhibitor will be administered in an amountsufficient to provide from about 1-150 mg per day.

In another embodiment, compounds of the invention are administered incombination with a dual-acting angiotensin-converting enzyme/neprilysin(ACE/NEP) inhibitor, examples of which include, but are not limited to:AVE-0848((4S,7S,12bR)-7-[3-methyl-2(S)-sulfanylbutyramido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]-benzazepine-4-carboxylicacid); AVE-7688 (ilepatril) and its parent compound; BMS-182657(2-[2-oxo-3(S)-[3-phenyl-2(S)-sulfanylpropionamido]-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]aceticacid); CGS-35601(N-[1-[4-methyl-2(S)-sulfanylpentanamido]cyclopentylcarbonyl]-L-tryptophan);fasidotril; fasidotrilate; enalaprilat; ER-32935((3R,6S,9aR)-6-[3(S)-methyl-2(S)-sulfanylpentanamido]-5-oxoperhydrothiazolo[3,2-a]azepine-3-carboxylicacid); gempatrilat; MDL-101264((4S,7S,12bR)-7-[2(S)-(2-morpholinoacetylthio)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); MDL-101287([4S-[4α,7α(R*),12bβ]]-7-[2-(carboxymethyl)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); omapatrilat; RB-105(N-[2(S)-(mercaptomethyl)-3(R)-phenylbutyl]-L-alanine); sampatrilat;SA-898((2R,4R)—N-[2-(2-hydroxyphenyl)-3-(3-mercaptopropionyl)thiazolidin-4-ylcarbonyl]-L-phenylalanine);Sch-50690(N-[1(S)-carboxy-2-[N2-(methanesulfonyl)-L-lysylamino]ethyl]-L-valyl-L-tyrosine);and combinations thereof, may also be included. In one particularembodiment, the ACE/NEP inhibitor is selected from: AVE-7688,enalaprilat, fasidotril, fasidotrilate, omapatrilat, sampatrilat, andcombinations thereof.

In one embodiment, compounds of the invention are administered incombination with an angiotensin-converting enzyme 2 (ACE2) activator orstimulator.

In one embodiment, compounds of the invention are administered incombination with an angiotensin-II vaccine, examples of which include,but are not limited to ATR12181 and CYT006-AngQb.

In one embodiment, compounds of the invention are administered incombination with an anticoagulant, representative examples of whichinclude, but are not limited to: coumarins such as warfarin; heparin;and direct thrombin inhibitors such as argatroban, bivalirudin,dabigatran, and lepirudin.

In yet another embodiment, compounds of the invention are administeredin combination with an anti-diabetic agent. Representative anti-diabeticagents include injectable drugs as well as orally effective drugs, andcombinations thereof. Examples of injectable drugs include, but are notlimited to, insulin and insulin derivatives. Examples of orallyeffective drugs include, but are not limited to: biguanides such asmetformin; glucagon antagonists; α-glucosidase inhibitors such asacarbose and miglitol; dipeptidyl peptidase IV inhibitors (DPP-IVinhibitors) such as alogliptin, denagliptin, linagliptin, saxagliptin,sitagliptin, and vildagliptin; meglitinides such as repaglinide;oxadiazolidinediones; sulfonylureas such as chlorpropamide, glimepiride,glipizide, glyburide, and tolazamide; thiazolidinediones such aspioglitazone and rosiglitazone; and combinations thereof.

In another embodiment, compounds of the invention are administered incombination with antidiarrheal treatments. Representative treatmentoptions include, but are not limited to, oral rehydration solutions(ORS), loperamide, diphenoxylate, and bismuth subsalicylate.

In yet another embodiment, a compound of the invention is administeredin combination with an anti-glaucoma agent. Representative anti-glaucomaagents include, but are not limited to: α-adrenergic agonists such asbrimonidine; β₁-adrenergic receptor antagonists; topical β₁-blockerssuch as betaxolol, levobunolol, and timolol; carbonic anhydraseinhibitors such as acetazolamide, brinzolamide, or dorzolamide;cholinergic agonists such as cevimeline and DMXB-anabaseine; epinephrinecompounds; miotics such as pilocarpine; and prostaglandin analogs.

In yet another embodiment, compounds of the invention are administeredin combination with an anti-lipid agent. Representative anti-lipidagents include, but are not limited to: cholesteryl ester transferprotein inhibitors (CETPs) such as anacetrapib, dalcetrapib, andtorcetrapib; statins such as atorvastatin, fluvastatin, lovastatin,pravastatin, rosuvastatin and simvastatin; and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an anti-thrombotic agent. Representativeanti-thrombotic agents include, but are not limited to: aspirin;anti-platelet agents such as clopidogrel, prasugrel, and ticlopidine;heparin, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an AT₁ receptor antagonist, also known as angiotensinII type 1 receptor blockers (ARBs). Representative ARBs include, but arenot limited to, abitesartan, azilsartan (e.g., azilsartan medoxomil),benzyllosartan, candesartan, candesartan cilexetil, elisartan,embusartan, enoltasosartan, eprosartan, EXP3174, fonsartan, forasartan,glycyllosartan, irbesartan, isoteoline, losartan, medoximil,milfasartan, olmesartan (e.g., olmesartan medoxomil), opomisartan,pratosartan, ripisartan, saprisartan, saralasin, sarmesin, TAK-591,tasosartan, telmisartan, valsartan, zolasartan, and combinationsthereof. In a particular embodiment, the ARB is selected from azilsartanmedoxomil, candesartan cilexetil, eprosartan, irbesartan, losartan,olmesartan medoxomil, saprisartan, tasosartan, telmisartan, valsartan,and combinations thereof. Exemplary salts and/or prodrugs includecandesartan cilexetil, eprosartan mesylate, losartan potassium salt, andolmesartan medoxomil. Typically, the ARB will be administered in anamount sufficient to provide from about 4-600 mg per dose, withexemplary daily dosages ranging from 20-320 mg per day.

Compounds of the invention may also be administered in combination witha dual-acting agent, such as an AT₁ receptor antagonist/neprilysininhibitor (ARB/NEP) inhibitor, examples of which include, but are notlimited to, compounds described in U.S. Publication Nos. 2008/0269305and 2009/0023228, both to Allegretti et al. filed on Apr. 23, 2008, suchas the compound,4′-{2-ethoxy-4-ethyl-5-[((S)-2-mercapto-4-methylpentanoylamino)-methyl]imidazol-1-ylmethyl}-3′-fluorobiphenyl-2-carboxylicacid.

Compounds of the invention may also be administered in combination withmultifunctional angiotensin receptor blockers as described in Kurtz &Klein (2009) Hypertension Research 32:826-834.

In one embodiment, compounds of the invention are administered incombination with a bradykinin receptor antagonist, for example,icatibant (HOE-140). It is expected that this combination therapy maypresent the advantage of preventing angioedema or other unwantedconsequences of elevated bradykinin levels.

In one embodiment, compounds of the invention are administered incombination with a calcium channel blocker. Representative calciumchannel blockers include, but are not limited to, amlodipine, anipamil,aranipine, barnidipine, bencyclane, benidipine, bepridil, clentiazem,cilnidipine, cinnarizine, diltiazem, efonidipine, elgodipine, etafenone,felodipine, fendiline, flunarizine, gallopamil, isradipine, lacidipine,lercanidipine, lidoflazine, lomerizine, manidipine, mibefradil,nicardipine, nifedipine, niguldipine, niludipine, nilvadipine,nimodipine, nisoldipine, nitrendipine, nivaldipine, perhexiline,prenylamine, ryosidine, semotiadil, terodiline, tiapamil, verapamil, andcombinations thereof. In a particular embodiment, the calcium channelblocker is selected from amlodipine, bepridil, diltiazem, felodipine,isradipine, lacidipine, nicardipine, nifedipine, niguldipine,niludipine, nimodipine, nisoldipine, ryosidine, verapamil, andcombinations thereof. Typically, the calcium channel blocker will beadministered in an amount sufficient to provide from about 2-500 mg perdose.

In one embodiment, compounds of the invention are administered incombination with a chymase inhibitor, such as TPC-806 and2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[{3,4-dioxo-1-phenyl-7-(2-pyridyloxy)}-2-heptyl]acetamide(NK3201).

In one embodiment, compounds of the invention are administered incombination with a diuretic. Representative diuretics include, but arenot limited to: carbonic anhydrase inhibitors such as acetazolamide anddichlorphenamide; loop diuretics, which include sulfonamide derivativessuch as acetazolamide, ambuside, azosemide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone, disulfamide,ethoxzolamide, furosemide, mefruside, methazolamide, piretanide,torsemide, tripamide, and xipamide, as well as non-sulfonamide diureticssuch as ethacrynic acid and other phenoxyacetic acid compounds such astienilic acid, indacrinone and quincarbate; osmotic diuretics such asmannitol; potassium-sparing diuretics, which include aldosteroneantagonists such as spironolactone, and Na⁺ channel inhibitors such asamiloride and triamterene; thiazide and thiazide-like diuretics such asalthiazide, bendroflumethiazide, benzylhydrochlorothiazide,benzthiazide, buthiazide, chlorthalidone, chlorothiazide,cyclopenthiazide, cyclothiazide, epithiazide, ethiazide, fenquizone,flumethiazide, hydrochlorothiazide, hydroflumethiazide, indapamide,methylclothiazide, meticrane, metolazone, paraflutizide, polythiazide,quinethazone, teclothiazide, and trichloromethiazide; and combinationsthereof. In a particular embodiment, the diuretic is selected fromamiloride, bumetanide, chlorothiazide, chlorthalidone, dichlorphenamide,ethacrynic acid, furosemide, hydrochlorothiazide, hydroflumethiazide,indapamide, methylclothiazide, metolazone, torsemide, triamterene, andcombinations thereof. The diuretic will be administered in an amountsufficient to provide from about 5-50 mg per day, more typically 6-25 mgper day, with common dosages being 6.25 mg, 12.5 mg or 25 mg per day.

Compounds of the invention may also be administered in combination withan endothelin converting enzyme (ECE) inhibitor, examples of whichinclude, but are not limited to, phosphoramidon, CGS 26303, andcombinations thereof.

In a particular embodiment, compounds of the invention are administeredin combination with an endothelin receptor antagonist. Representativeendothelin receptor antagonists include, but are not limited to:selective endothelin receptor antagonists that affect endothelin Areceptors, such as avosentan, ambrisentan, atrasentan, BQ-123,clazosentan, darusentan, sitaxentan, and zibotentan; and dual endothelinreceptor antagonists that affect both endothelin A and B receptors, suchas bosentan, macitentan, tezosentan).

In yet another embodiment, a compound of the invention is administeredin combination with one or more HMG-CoA reductase inhibitors, which arealso known as statins. Representative statins include, but are notlimited to, atorvastatin, fluvastatin, lovastatin, pitavastatin,pravastatin, rosuvastatin and simvastatin.

In one embodiment, compounds of the invention are administered incombination with a monoamine reuptake inhibitor, examples of whichinclude, by way of illustration and not limitation, norepinephrinereuptake inhibitors such as atomoxetine, buproprion and the buproprionmetabolite hydroxybuproprion, maprotiline, reboxetine, and viloxazine;selective serotonin reuptake inhibitors (SSRIs) such as citalopram andthe citalopram metabolite desmethylcitalopram, dapoxetine, escitalopram(e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethylmetabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate),paroxetine, sertraline and the sertraline metabolite demethylsertraline;dual serotonin-norepinephrine reuptake inhibitors (SNRIs) such asbicifadine, duloxetine, milnacipran, nefazodone, and venlafaxine; andcombinations thereof.

In another embodiment, compounds of the invention are administered incombination with a muscle relaxant, examples of which include, but arenot limited to: carisoprodol, chlorzoxazone, cyclobenzaprine,diflunisal, metaxalone, methocarbamol, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a natriuretic peptide or analog, examples of whichinclude but are not limited to: carperitide, CD-NP (Nile Therapeutics),CU-NP, nesiritide, PL-3994 (Palatin Technologies, Inc.), ularitide,cenderitide, and compounds described in Ogawa et al (2004) J. Biol.Chem. 279:28625-31. These compounds are also referred to as natriureticpeptide receptor-A (NPR-A) agonists. In another embodiment, compounds ofthe invention are administered in combination with a natriuretic peptideclearance receptor (NPR-C) antagonist such as SC-46542, cANF (4-23), andAP-811 (Veale (2000) Bioorg Med Chem Lett 10:1949-52). For example,AP-811 has shown synergy when combined with the NEP inhibitor, thiorphan(Wegner (1995) Clin. Exper. Hypert. 17:861-876).

In another embodiment, compounds of the invention are administered incombination with a neprilysin (NEP) inhibitor. Representative NEPinhibitors include, but are not limited to: AHU-377; candoxatril;candoxatrilat; dexecadotril((+)-N-[2(R)-(acetylthiomethyl)-3-phenylpropionyl]glycine benzyl ester);CGS-24128(3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-24592((S)-3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-25155(N-[9(R)-(acetylthiomethyl)-10-oxo-1-azacyclodecan-2(S)-ylcarbonyl]-4(R)-hydroxy-L-prolinebenzyl ester); 3-(1-carbamoylcyclohexyl)propionic acid derivativesdescribed in WO 2006/027680 to Hepworth et al. (Pfizer Inc.); JMV-390-1(2(R)-benzyl-3-(N-hydroxycarbamoyl)propionyl-L-isoleucyl-L-leucine);ecadotril; phosphoramidon; retrothiorphan; RU-42827(2-(mercaptomethyl)-N-(4-pyridinyl)benzenepropionamide); RU-44004(N-(4-morpholinyl)-3-phenyl-2-(sulfanylmethyl)propionamide); SCH-32615((S)—N—[N-(1-carboxy-2-phenylethyl)-L-phenylalanyl]-β-alanine) and itsprodrug SCH-34826((S)—N—[N-[1-[[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β-alanine);sialorphin; SCH-42495(N-[2(S)-(acetylsulfanylmethyl)-3-(2-methylphenyl)propionyl]-L-methionineethyl ester); spinorphin; SQ-28132(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]leucine); SQ-28603(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-β-alanine); SQ-29072(7-[[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid);thiorphan and its prodrug racecadotril; UK-69578(cis-4-[[[1-[2-carboxy-3-(2-methoxyethoxyl)propyl]cyclopentyl]carbonyl]amino]cyclohexanecarboxylicacid); UK-447,841(2-{1-[3-(4-chlorophenyl)propylcarbamoyl]-cyclopentylmethyl}-4-methoxybutyricacid); UK-505,749((R)-2-methyl-3-{1-[3-(2-methylbenzothiazol-6-yl)propylcarbamoyl]cyclopentyl}propionicacid); 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid and 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid ethyl ester (WO 2007/056546); daglutril[(3S,2′R)-3-{1-[2′-(ethoxycarbonyl)-4′-phenylbutyl]-cyclopentan-1-carbonylamino}-2,3,4,5-tetrahydro-2-oxo-1H-1-benzazepine-1-aceticacid] described in WO 2007/106708 to Khder et al. (Novartis AG); andcombinations thereof. In a particular embodiment, the NEP inhibitor isselected from AHU-377, candoxatril, candoxatrilat, CGS-24128,phosphoramidon, SCH-32615, SCH-34826, SQ-28603, thiorphan, andcombinations thereof. In a particular embodiment, the NEP inhibitor is acompound such as daglutril or CGS-26303([N-[2-(biphenyl-4-yl)-1(S)-(1H-tetrazol-5-yl)ethyl]amino]methylphosphonicacid), which have activity both as inhibitors of the endothelinconverting enzyme (ECE) and of NEP. Other dual acting ECE/NEP compoundscan also be used. The NEP inhibitor will be administered in an amountsufficient to provide from about 20-800 mg per day, with typical dailydosages ranging from 50-700 mg per day, more commonly 100-600 or 100-300mg per day.

In one embodiment, compounds of the invention are administered incombination with a nitric oxide donor, examples of which include, butare not limited to nicorandil; organic nitrates such as pentaerythritoltetranitrate; and sydnonimines such as linsidomine and molsidomine.

In yet another embodiment, compounds of the invention are administeredin combination with a non-steroidal anti-inflammatory agent (NSAID).Representative NSAIDs include, but are not limited to: acemetacin,acetyl salicylic acid, alclofenac, alminoprofen, amfenac, amiprilose,aloxiprin, anirolac, apazone, azapropazone, benorilate, benoxaprofen,bezpiperylon, broperamole, bucloxic acid, carprofen, clidanac,diclofenac, diflunisal, diftalone, enolicam, etodolac, etoricoxib,fenbufen, fenclofenac, fenclozic acid, fenoprofen, fentiazac, feprazone,flufenamic acid, flufenisal, fluprofen, flurbiprofen, furofenac,ibufenac, ibuprofen, indomethacin, indoprofen, isoxepac, isoxicam,ketoprofen, ketorolac, lofemizole, lornoxicam, meclofenamate,meclofenamic acid, mefenamic acid, meloxicam, mesalamine, miroprofen,mofebutazone, nabumetone, naproxen, niflumic acid, oxaprozin, oxpinac,oxyphenbutazone, phenylbutazone, piroxicam, pirprofen, pranoprofen,salsalate, sudoxicam, sulfasalazine, sulindac, suprofen, tenoxicam,tiopinac, tiaprofenic acid, tioxaprofen, tolfenamic acid, tolmetin,triflumidate, zidometacin, zomepirac, and combinations thereof. In aparticular embodiment, the NSAID is selected from etodolac,flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meloxicam,naproxen, oxaprozin, piroxicam, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an N-methyl d-aspartate (NMDA) receptor antagonist,examples of which include, by way of illustration and not limitation,including amantadine, dextromethorphan, dextropropoxyphene, ketamine,ketobemidone, memantine, methadone, and so forth.

In still another embodiment, compounds of the invention are administeredin combination with an opioid receptor agonist (also referred to asopioid analgesics). Representative opioid receptor agonists include, butare not limited to: buprenorphine, butorphanol, codeine, dihydrocodeine,fentanyl, hydrocodone, hydromorphone, levallorphan, levorphanol,meperidine, methadone, morphine, nalbuphine, nalmefene, nalorphine,naloxone, naltrexone, nalorphine, oxycodone, oxymorphone, pentazocine,propoxyphene, tramadol, and combinations thereof. In certainembodiments, the opioid receptor agonist is selected from codeine,dihydrocodeine, hydrocodone, hydromorphone, morphine, oxycodone,oxymorphone, tramadol, and combinations thereof.

In a particular embodiment, compounds of the invention are administeredin combination with a phosphodiesterase (PDE) inhibitor, particularly aPDE-V inhibitor. Representative PDE-V inhibitors include, but are notlimited to, avanafil, lodenafil, mirodenafil, sildenafil (Revatio®),tadalafil (Adcirca®), vardenafil (Levitra®), and udenafil.

In another embodiment, compounds of the invention are administered incombination with a prostaglandin analog (also referred to as prostanoidsor prostacyclin analogs). Representative prostaglandin analogs include,but are not limited to, beraprost sodium, bimatoprost, epoprostenol,iloprost, latanoprost, tafluprost, travoprost, and treprostinil, withbimatoprost, latanoprost, and tafluprost being of particular interest.

In yet another embodiment, compounds of the invention are administeredin combination with a prostaglandin receptor agonist, examples of whichinclude, but are not limited to, bimatoprost, latanoprost, travoprost,and so forth.

Compounds of the invention may also be administered in combination witha renin inhibitor, examples of which include, but are not limited to,aliskiren, enalkiren, remikiren, and combinations thereof.

In another embodiment, compounds of the invention are administered incombination with a selective serotonin reuptake inhibitor (SSRI).Representative SSRIs include, but are not limited to: citalopram and thecitalopram metabolite desmethylcitalopram, dapoxetine, escitalopram(e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethylmetabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate),paroxetine, sertraline and the sertraline metabolite demethylsertraline,and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a 5-HT_(1D) serotonin receptor agonist, examples ofwhich include, by way of illustration and not limitation, triptans suchas almotriptan, avitriptan, eletriptan, frovatriptan, naratriptanrizatriptan, sumatriptan, and zolmitriptan.

In one embodiment, compounds of the invention are administered incombination with a sodium channel blocker, examples of which include, byway of illustration and not limitation, carbamazepine, fosphenytoin,lamotrigine, lidocaine, mexiletine, oxcarbazepine, phenytoin, andcombinations thereof.

In one embodiment, compounds of the invention are administered incombination with a soluble guanylate cyclase stimulator or activator,examples of which include, but are not limited to ataciguat, riociguat,and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a tricyclic antidepressant (TCA), examples of whichinclude, by way of illustration and not limitation, amitriptyline,amitriptylinoxide, butriptyline, clomipramine, demexiptiline,desipramine, dibenzepin, dimetacrine, dosulepin, doxepin, imipramine,imipraminoxide, lofepramine, melitracen, metapramine, nitroxazepine,nortriptyline, noxiptiline, pipofezine, propizepine, protriptyline,quinupramine, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a vasopressin receptor antagonist, examples of whichinclude, by way of illustration and not limitation, conivaptan andtolvaptan.

Combined secondary therapeutic agents may also be helpful in furthercombination therapy with compounds of the invention. For example,compounds of the invention can be combined with a diuretic and an ARB,or a calcium channel blocker and an ARB, or a diuretic and an ACEinhibitor, or a calcium channel blocker and a statin. Specific examplesinclude, a combination of the ACE inhibitor enalapril (in the maleatesalt form) and the diuretic hydrochlorothiazide, which is sold under themark Vaseretic®, or a combination of the calcium channel blockeramlodipine (in the besylate salt form) and the ARB olmesartan (in themedoxomil prodrug form), or a combination of a calcium channel blockerand a statin, all may also be used with the compounds of the invention.Other therapeutic agents such as α₂-adrenergic receptor agonists andvasopressin receptor antagonists may also be helpful in combinationtherapy. Exemplary α₂-adrenergic receptor agonists include clonidine,dexmedetomidine, and guanfacine.

The following formulations illustrate representative pharmaceuticalcompositions of the invention.

Exemplary Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), 440 g spray-dried lactose and 10 gmagnesium stearate are thoroughly blended. The resulting composition isthen loaded into hard gelatin capsules (500 mg of composition percapsule). Alternately, a compound of the invention (20 mg) is thoroughlyblended with starch (89 mg), microcrystalline cellulose (89 mg) andmagnesium stearate (2 mg). The mixture is then passed through a No. 45mesh U.S. sieve and loaded into a hard gelatin capsule (200 mg ofcomposition per capsule).

Alternately, a compound of the invention (30 g), a secondary agent (20g), 440 g spray-dried lactose and 10 g magnesium stearate are thoroughlyblended, and processed as described above.

Exemplary Gelatin Capsule Formulation for Oral Administration

A compound of the invention (100 mg) is thoroughly blended withpolyoxyethylene sorbitan monooleate (50 mg) and starch powder (250 mg).The mixture is then loaded into a gelatin capsule (400 mg of compositionper capsule). Alternately, a compound of the invention (70 mg) and asecondary agent (30 mg) are thoroughly blended with polyoxyethylenesorbitan monooleate (50 mg) and starch powder (250 mg), and theresulting mixture loaded into a gelatin capsule (400 mg of compositionper capsule).

Alternately, a compound of the invention (40 mg) is thoroughly blendedwith microcrystalline cellulose (Avicel PH 103; 259.2 mg) and magnesiumstearate (0.8 mg). The mixture is then loaded into a gelatin capsule(Size #1, White, Opaque) (300 mg of composition per capsule).

Exemplary Tablet Formulation for Oral Administration

A compound of the invention (10 mg), starch (45 mg) and microcrystallinecellulose (35 mg) are passed through a No. 20 mesh U.S. sieve and mixedthoroughly. The granules so produced are dried at 50-60° C. and passedthrough a No. 16 mesh U.S. sieve. A solution of polyvinylpyrrolidone (4mg as a 10% solution in sterile water) is mixed with sodiumcarboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg), and talc (1mg), and this mixture is then passed through a No. 16 mesh U.S. sieve.The sodium carboxymethyl starch, magnesium stearate and talc are thenadded to the granules. After mixing, the mixture is compressed on atablet machine to afford a tablet weighing 100 mg.

Alternately, a compound of the invention (250 mg) is thoroughly blendedwith microcrystalline cellulose (400 mg), silicon dioxide fumed (10 mg),and stearic acid (5 mg). The mixture is then compressed to form tablets(665 mg of composition per tablet).

Alternately, a compound of the invention (400 mg) is thoroughly blendedwith cornstarch (50 mg), croscarmellose sodium (25 mg), lactose (120mg), and magnesium stearate (5 mg). The mixture is then compressed toform a single-scored tablet (600 mg of composition per tablet).

Alternately, a compound of the invention (100 mg) is thoroughly blendedwith cornstarch (100 mg) with an aqueous solution of gelatin (20 mg).The mixture is dried and ground to a fine powder. Microcrystallinecellulose (50 mg) and magnesium stearate (5 mg) are then admixed withthe gelatin formulation, granulated and the resulting mixture compressedto form tablets (100 mg of the compound of the invention per tablet).

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of the compound of the invention per 10 mL of suspension:

Ingredients Amount Compound of the invention  1.0 g Fumaric acid  0.5 gSodium chloride  2.0 g Methyl paraben  0.15 g Propyl paraben  0.05 gGranulated sugar  25.5 g Sorbitol (70% solution) 12.85 g Veegum ® K  1.0g (magnesium aluminum silicate) Flavoring 0.035 mL Colorings  0.5 mgDistilled water q.s. to 100 mL

Exemplary Liquid Formulation for Oral Administration

A suitable liquid formulation is one with a carboxylic acid-based buffersuch as citrate, lactate and maleate buffer solutions. For example, acompound of the invention (which may be pre-mixed with DMSO) is blendedwith a 100 mM ammonium citrate buffer and the pH adjusted to pH 5, or isblended with a 100 mM citric acid solution and the pH adjusted to pH 2.Such solutions may also include a solubilizing excipient such as acyclodextrin, for example the solution may include 10 wt %hydroxypropyl-β-cyclodextrin.

Other suitable formulations include a 5% NaHCO₃ solution, with orwithout cyclodextrin.

Exemplary Injectable Formulation for Administration by Injection

A compound of the invention (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH of the resulting solution is adjustedto pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodiumhydroxide, as necessary, and then sufficient water for injection isadded to provide a total volume of 20 mL. The mixture is then filteredthrough a sterile filter (0.22 micron) to provide a sterile solutionsuitable for administration by injection.

Exemplary Compositions For Administration By Inhalation

A compound of the invention (0.2 mg) is micronized and then blended withlactose (25 mg). This blended mixture is then loaded into a gelatininhalation cartridge. The contents of the cartridge are administeredusing a dry powder inhaler, for example.

Alternately, a micronized compound of the invention (10 g) is dispersedin a solution prepared by dissolving lecithin (0.2 g) in demineralizedwater (200 mL). The resulting suspension is spray dried and thenmicronized to form a micronized composition comprising particles havinga mean diameter less than about 1.5 μm. The micronized composition isthen loaded into metered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μgto about 500 μg of the compound of the invention per dose whenadministered by the inhaler.

Alternately, a compound of the invention (25 mg) is dissolved in citratebuffered (pH 5) isotonic saline (125 mL). The mixture is stirred andsonicated until the compound is dissolved. The pH of the solution ischecked and adjusted, if necessary, to pH 5 by slowly adding aqueous 1 NNaOH. The solution is administered using a nebulizer device thatprovides about 10 μg to about 500 μg of the compound of the inventionper dose.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard, generally accepted meaning:

-   -   AcOH acetic acid    -   BOC t-butoxycarbonyl (—C(O)OC(CH₃)₃)    -   (BOC)₂O di-t-butyl dicarbonate    -   Bzl benzyl    -   DCC dicyclohexylcarbodiimide    -   DCM dichloromethane or methylene chloride    -   DIPEA N,N-diisopropylethylamine    -   DMAP 4-dimethylaminopyridine    -   DMF N,N-dimethylformamide    -   DMSO dimethyl sulfoxide    -   Et₃N triethylamine    -   Et₂O diethyl ether    -   EtOAc ethyl acetate    -   EtOH ethanol    -   HATU N,N,′N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hexafluorophosphate    -   LiHMDS lithium hexamethyl disilazide    -   MeCN acetonitrile    -   MeOH methanol    -   MTBE methyl t-butyl ether    -   NaHMDS sodium hexamethyldisilazide    -   Pd(dppf)₂Cl₂ 1,1-bis(diphenylphosphino) ferrocene palladium        chloride    -   PE petroleum ether (better known as hexanes)    -   TFA trifluoroacetic acid    -   THF tetrahydrofuran

Unless noted otherwise, all materials, such as reagents, startingmaterials and solvents, were purchased from commercial suppliers (suchas Sigma-Aldrich, Fluka Riedel-de Haën, and the like) and were usedwithout further purification.

Reactions were run under nitrogen atmosphere, unless noted otherwise.The progress of reactions were monitored by thin layer chromatography(TLC), analytical high performance liquid chromatography (anal. HPLC),and mass spectrometry, the details of which are given in specificexamples. Solvents used in analytical HPLC were as follows: solvent Awas 98% H₂O/2% MeCN/1.0 mL/L TFA; solvent B was 90% MeCN/10% H₂O/1.0mL/L TFA.

Reactions were worked up as described specifically in each preparationfor example; commonly reaction mixtures were purified by extraction andother purification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by preparative HPLC, typically using Microsorb C18and Microsorb BDS column packings and conventional eluents. Progress ofreactions was typically measured by liquid chromatography massspectrometry (LCMS). Characterization of isomers were done by NuclearOverhauser effect spectroscopy (NOE). Characterization of reactionproducts was routinely carried out by mass and ¹H-NMR spectrometry. ForNMR measurement, samples were dissolved in deuterated solvent (CD₃OD,CDCl₃, or DMSO-d₆), and ¹H-NMR spectra were acquired with a VarianGemini 2000 instrument (400 MHz) under standard observation conditions.Mass spectrometric identification of compounds was typically conductedusing an electrospray ionization method (ESMS) with an AppliedBiosystems (Foster City, Calif.) model API 150 EX instrument or anAgilent (Palo Alto, Calif.) model 1200 LC/MSD instrument.

Preparation 1:Oxodiperoxymolybdenum(pyridine)(hexamethylphosphorictriamide)

MoO₃→MoO₅.H₂O.(Me₂N)₃PO→MoO₅.(Me₂N)₃PO→MoO₅.Py.(Me₂N)₃PO

Molybdenum oxide (MoO₃; 30 g, 0.2 mol) and 30% hydrogen peroxide (150mL) were combined, with stirring. The reaction vessel was placed in anoil bath equilibrated at 40° C. and heated until the internaltemperature reached 35° C. The heating bath was then removed andreplaced by a water bath to control the mildly exothermic reaction sothat an internal temperature of 35-40° C. was maintained. After theinitial exothermic period (˜30 minutes), the reaction vessel wasreturned to the 40° C. oil bath and stirred for a total of 3.5 hours toform a yellow solution with a small amount of suspended white solid.After cooling to 20° C., the solution was filtered and the resultingyellow filtrate was cooled to 10° C. (ice bath with stirring) andhexamethylphosphoric triamide ((Me₂N)₃PO; HMPA; 37.3 g, 0.2 mol) wasadded dropwise over 5 minutes, resulting in the formation of a yellowcrystalline precipitate. Stirring was continued for a total of 15minutes at 10° C., and the product was filtered and pressed dry. After30 minutes under vacuum, the filter cake was combined with MeOH (20 mL)and stirred at 40° C. Additional MeOH was slowly added until the solidsdissolved. The saturated solution was cooled in the refrigerator,yielding a yellow solid (appeared as needles). The solid mass wasphysically broken, filtered and washed with cold MeOH (20-30 mL) toyield oxodiperoxymolybdenum(aqua) (hexamethylphosphoric triamide)(MoO₅.H₂O.HMPA, 46-50 g).

MoO₅.H₂O.HMPA was dried over phosphorus oxide in a vacuum desiccator,shielded from the light, for 24 hours at 0.2 mm Hg to yield a somewhathygroscopic yellow solid, MoO₅.HMPA. MoO5.HMPA (36.0 g, 0.1 mol) wasdissolved in THF (150 mL) and the solution was filtered to remove anyprecipitate. The filtrate was then stirred at 20° C. while dry pyridine(8.0 g, 0.1 mol) was added over 10 minutes. The crystalline, yellowproduct was collected, washed with dry THF (25 mL) and anhydrous ether(200 mL) and dried in a vacuum desiccator (1 hour, 0.2 mm Hg) to yieldthe title compound, oxodiperoxymolybdenum(pyridine)(hexamethylphosphorictriamide) (MoO₅.Py.HMPA) as a finely divided yellow solid (36-38 g).

Preparation 2: (2R,4R)-4-Amino-5-biphenyl-4-yl-2-hydroxypentanoic AcidEthyl Ester (Compound 7) and(2R,4S)-4-Amino-5-biphenyl-4-yl-2-hydroxypentanoic Acid Ethyl Ester(Compound 9)

To a stirred solution of(R)-3-biphenyl-4-yl-2-t-butoxycarbonylaminopropionic acid (50 g, 146.5mmol), Meldrum's acid (23.3 g, 161.1 mmol) and DMAP (27.8 g, 227 mmol)in anhydrous DCM (500 mL) was added a solution of DCC (33.3 g, 161.1mmol) in anhydrous DCM (200 mL) over 1 hour at −5° C. under nitrogen.The mixture was stirred at −5° C. for 8 hours, then refrigeratedovernight, during which tiny crystals of dicyclohexylurea precipitated.After filtration the mixture was washed with 5% KHSO₄ (4×200 mL),saturated aqueous NaCl (1×200 mL) and dried over MgSO₄ overnight. Theresulting solution was evaporated to give the crude Compound 1 (68 g) asa light yellow solid). LC-MS: [M⁺Na]: 490, [2M⁺Na]: 957.

To a solution of crude Compound 1 (68 g, 146.5 mmol) in anhydrous DCM(1000 mL) was added AcOH (96.8 g, 1.6 mol) at −5° C. under nitrogen. Theresulting mixture was stirred at −5° C. for 0.5 hours, then NaBH₄ (13.9g, 366 mmol) was added in small portions over 2 hours. After stirringfor another hour at −5° C., saturated aqueous NaCl (300 mL) was added.The organic layer was washed with saturated aqueous NaCl (2×300 mL) thenwater (2×300 mL), dried over MgSO₄, filtered, and evaporated to give thecrude Compound 2, which was further purified by chromatography(hexanes:EtOAc=5:1) to give purified Compound 2 (46 g) as a light yellowsolid. LC-MS: [M⁺Na]: 476, [2M⁺Na]: 929.

A stirred solution of purified Compound 2 (46 g, 101 mmol) in anhydroustoluene (300 mL) was heated to reflux under nitrogen for 3 hours. Afterevaporation of the solvent, the residue was purified by chromatography(hexanes:EtOAc=10:1) to yield Compound 3 (27 g) as a light yellow solid.

LC-MS: [M⁺Na]: 374, [2M⁺Na]: 725; 1H NMR (300 MHz, CDCl₃): δ7.64-7.62(m, 4H), 7.51-7.46 (m, 2H), 7.42-7.39 (m, 1H), 7.39-7.30 (m, 2H),4.50-4.43 (m, 1H), 3.27-3.89 (m, 1H), 2.88-2.80 (m, 1H), 2.48-2.42 (m,2H), 2.09-1.88 (m, 2H), 1.66 (s, 9H).

To a stirred solution of Compound 3 (4.4 g, 12.4 mmol) in anhydrous THF(70 mL) was added a solution of 1 M LiHMDS in THF (28 mL) over 15minutes at −65° C. under nitrogen. After stirring for 3 hours at −65°C., oxodiperoxymolybdenum(pyridine)(hexamethylphosphorictriamide) (9 g,18.6 mmol) was added. The mixture was stirred for another 2 hours at−35° C., then saturated aqueous Na₂S₂O₃ (60 mL) was added. The organiclayer was collected and washed with saturated aqueous NH₄Cl (60 mL×3)and saturated aqueous NaCl (60 mL×2), then dried over Na₂SO₄, and thesolvent was removed under reduced pressure to yield the crude productwhich was further purified by chromatography (hexanes:EtOAc=5:1) toyield Compound 4 as a white solid (1.8 g). LC-MS: [2M+Na]: 757.

To a solution of Compound 4 (1.8 g, 5.0 mmol) in anhydrous DCM (50 mL)was added DMAP (122 mg, 1 mmol) and Et₃N (1.5 g, 14.9 mmol) at 0° C.under nitrogen. After stirring for 0.5 hour at 0° C., benzyl chloride(1.0 g, 7.4 mmol) was added over 15 minutes. The mixture was stirred foran additional 2 hours at 0° C., then saturated aqueous NaHCO₃ (50 mL)was added. The organic layer was collected and washed with saturatedaqueous NaHCO₃ (50 mL×2) and saturated aqueous NaCl (50 mL×1), thendried over Na₂SO₄. The solids were filtered out and the filtrate wasconcentrated to yield the crude product which was further purified bychromatography (hexanes:EtOAc=4:1) to yield Compound 5A (471 mg) andCompound 5B (883 mg) as white solids. LC-MS: [M+Na]: 494; [2M+Na]: 965.

Compound 5A: ¹H NMR (300 MHz, CDCl₃): δ (ppm)=8.02 (m, 2H), 7.57-7.25(m, 12H), 5.42 (m, 1H), 4.50 (m, 1H), 3.26-3.21 (m, 1H), 2.90 (m, 1H),2.58 (m, 1H), 2.15-2.05 (m, 1H), 1.62 (m, 9H)

Compound 5B: ¹H NMR (300 MHz, CDCl₃): δ (ppm)=8.06 (m, 2H), 7.58-7.18(m, 12H), 5.53-5.41 (m, 1H), 4.39 (m, 1H), 3.57-3.54 (m, 1H), 2.87-2.80(m, 1H), 2.48-2.44 (m, 1H), 1.98 (m, 1H), 1.63 (m, 9H).

To a stirred solution of Compound 5A (471 mg, 1 mmol) in anhydrous EtOH(10 mL) was added anhydrous K₂CO₃ (691 mg, 5 mmol) at room temperatureunder nitrogen. After stirring for 20 hours at room temperature, thesolids were filtered out. To the filtrate was added water (30 mL), DCM(30 mL) and saturated aqueous NaCl (5 mL). The aqueous layer wasseparated and extracted with DCM (30 mL×3). The combined organic layerswere washed with saturated aqueous NaCl (50 mL), dried over Na₂SO₄, andconcentrated to yield the crude product which was further purified bychromatography (hexanes:EtOAc=6:1) to yield Compound 6 as a white solid(275 mg). LC-MS: [M+Na]: 436, [2M+Na]: 849.

To EtOH (5 mL) was added acetyl chloride (685 mg) at −30° C. Afterstirring for 1 hour at −30° C., a solution of Compound 6 (275 mg, 665μmol) in anhydrous EtOH (5 mL) was added. The mixture was heated to 25°C. and stirred for 3 hours at 25° C. After evaporation of the solvent,the residue was washed with cold anhydrous Et₂O (10 mL) to yieldCompound 7 as a white solid HCl salt (207 mg). LC-MS: [M+H]: 314,[2M+Na]: 649.

¹H NMR (300 MHz, CDCl₃): δ (ppm)=7.99 (m, 3H), 7.66-7.64 (m, 4H),7.48-7.35 (m, 5H), 6.08 (m, 1H), 4.21 (m, 1H), 4.09-4.05 (m, 2H), 3.52(m, 1H), 2.97-2.95 (m, 2H), 1.89-1.87 (m, 2H), 1.19-1.14 (m, 3H).

To a stirred solution of Compound 5B (883 mg, 1.9 mmol) in anhydrousEtOH (15 mL) was added anhydrous K₂CO₃ (1293 mg, 9.4 mmol) at roomtemperature under nitrogen. After stirring for 20 hours at roomtemperature, the solids were filtered out. To the filtrate was addedwater (30 mL), DCM (30 mL) and saturated aqueous NaCl (5 mL). Theaqueous layer was separated and extracted with DCM (30 mL×3). Thecombined organic layers were washed with saturated aqueous NaCl (50 mL),dried over Na₂SO₄, and concentrated to yield the crude product which wasfurther purified by chromatography (hexanes:EtOAc=6:1) to yield Compound8 as a white solid (524 mg). LC-MS: [M+Na]: 436, [2M+Na]: 849.

To EtOH (8 mL) was added acetyl chloride (1300 mg) at −30° C. Afterstirring for 1 hour at −30° C., a solution of Compound 8 (524 mg, 1.3mmol) in anhydrous EtOH (8 mL) was added. The mixture was heated to 25°C. and stirred for 3 hours at 25° C. After evaporation of the solvent,the residue was washed with cold anhydrous Et₂O (10 mL) to yieldCompound 9 as a white solid HCl salt (395 mg). LC-MS: [M+H]: 314,[2M+Na]: 649.

¹H NMR (300 MHz, CDCl₃): δ (ppm)=8.14 (m, 3H), 7.66-7.62 (m, 4H),7.47-7.31 (m, 5H), 5.87-5.85 (m, 1H), 4.34 (m, 1H), 4.08-4.00 (m, 2H),3.48 (m, 1H), 3.09 (m, 1H), 2.85-2.81 (m, 1H), 1.88 (m, 1H), 1.76 (m,1H), 1.15-1.10 (m, 3H).

Preparation 3: (S)-2-(4-Bromobenzyl)-5-oxopyrrolidine-1-carboxylic Acidt-Butyl Ester

To a solution of (R)-2-amino-3-(4-bromophenyl)propionic acid (50 g, 0.2mol) in MeCN (700 mL) was added a solution of NaOH (16.4 g, 0.4 mol) inwater (700 mL) at −5° C. After stirring for 10 minutes, a solution of(BOC)₂O (44.7 g, 0.2 mol) in MeCN (100 mL) was added. The mixture waswarmed to room temperature and stirred overnight. After the evaporationof the MeCN, the residue was diluted with DCM (800 mL) and acidifiedwith 1 M HCl to pH 2 at −5° C. The aqueous was extracted with DCM (3×200mL). The combined organic layers were washed with saturated aqueous NaCl(500 mL), dried over Na₂SO₄ and concentrated to yield Compound 1 (66.5g) as a white solid. LC-MS: 366 (M+Na), 709 (2M+Na).

To a solution of Compound 1 (66.5 g, 193 μmol), Meldrum's acid (33.4 g,232 mmol) and DMAP (37.7 g, 309 mmol) in anhydrous DCM (600 mL), wasadded dropwise a solution of DCC (47.9 g, 232 mmol) in anhydrous DCM(200 mL) over 1 hour at −5° C. under nitrogen. The mixture was stirredat −5° C. for 8 hours, then refrigerated overnight. Crystals ofdicyclohexylurea were observed. The mixture was filtered, washed with 5%KHSO₄ (5×200 mL) and saturated aqueous NaCl (200 mL), then dried overanhydrous MgSO₄ under refrigeration overnight. The solution was thenevaporated to yield crude Compound 2 (91 g) as a light yellow solid.LC-MS: 492 (M+Na), 961 (2M+Na).

To a solution of crude Compound 2 (91 g, 193 mmol) in anhydrous DCM (1L) was added AcOH (127.5 g, 2.1 mol) at −5° C. under nitrogen. Themixture was stirred at −5° C. for 30 minutes, then NaBH₄ (18.3 g, 483mmol) was added in small portions over 1 hour. After stirring foranother 1 hour at −5° C., saturated aqueous NaCl (500 mL) was added. Theorganic layer was washed with saturated aqueous NaCl (2×300 mL) andwater (2×300 mL), dried over MgSO₄, filtered, and concentrated to yieldthe crude product, which was further purified by washing with Et₂O toyield Compound 3 (68 g) as a light yellow solid. LC-MS: 478 (M+Na), 933(2M+Na).

A solution of Compound 3 (68 g, 149 mmol) in anhydrous toluene (500 mL)was refluxed under nitrogen for 3 hours. After evaporation of thesolvent, the residue was purified by chromatography (hexanes:EtOAc=10:1)to yield the title compound (38 g) as a light yellow oil. LC-MS: 376(M+Na), 729 (2M+Na).

Preparation 4:(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic Acid EthylEster

To a solution of (S)-2-(4-bromobenzyl)-5-oxopyrrolidine-1-carboxylicacid t-butyl ester (15 g, 43 mmol) in 1,4-dioxane (600 mL) was added3-chlorophenylboronic acid (8 g, 51 mmol) and Pd(dppf)₂Cl₂ (3.1 g, 4.2mmol) at room temperature under nitrogen. After stirring for 10 minutes,a solution of K₂CO₃ (11.7 g, 85 mmol) in water (60 mL) was added. Themixture was heated to 60° C. and stirred overnight. After evaporation ofthe solvent, water (200 mL) was added and extracted with EtOAc (3×200mL). The combined organic layers were washed with saturated aqueous NaCl(400 mL), dried over Na₂SO₄, and concentrated to yield the crude productwhich was further purified by column chromatography (hexanes:EtOAc=6:1)to yield Compound 1 (15 g) as a light yellow solid. LC-MS: 408 (M+Na).

To a solution of Compound 1 (15 g, 0.039 mol) in anhydrous DCM (250 mL)was added TFA (20 mL, 270 mmol) at −5° C. under nitrogen. The mixturewas warmed to room temperature and stirred overnight. After evaporationof the solvent, the residue was diluted with EtOAc (300 mL), then washedwith saturated aqueous NaHCO₃ (3×200 mL), water (200 mL), and saturatedaqueous NaCl (250 mL), then dried over Na₂SO₄ and concentrated to yieldcrude Compound 2 (11 g) as a light yellow solid. LC-MS: 286 [M+H].

To a solution of NaH (2.3 g, 98 mmol) in anhydrous THF (200 mL) wasadded dropwise a solution of Compound 2 (11 g, 39 mmol) in anhydrous THF(100 mL) over 30 minutes at 0° C. under nitrogen. The mixture was warmedto room temperature and stirred for 2 hours. After cooling to 0° C.,pivaloyl chloride (6 g, 51 mmol) was added dropwise over 30 minutes. Themixture was warmed to room temperature and stirred overnight. Thereaction was quenched with saturated aqueous NH₄Cl (200 mL) andextracted with EtOAc (3×200 mL). The combined organic layers were washedwith saturated aqueous NaCl (300 mL), dried over MgSO₄, filtered, andconcentrated to yield the crude product which was further purified bychromatography (hexanes:EtOAc=25:1) to yield Compound 3 (10.5 g) as alight yellow solid. LC-MS: 391 (M+Na).

To a solution of Compound 3 (10.5 g, 29 mmol) in anhydrous THF (120 mL)was added dropwise NaHMDS (29 mL, 58 mmol) over 30 minutes at −78° C.under nitrogen. After stirring at −78° C. for 90 minutes, a solution of(+)-(8,8-dichlorocamphorylsulfonyl)-oxaziridine (15.6 g, 52 mmol) wasadded dropwise over 30 minutes. After stirring at −78° C. for 2 hours,the reaction was quenched with saturated NH₄Cl (400 mL) and extractedwith EtOAc (3×300 mL). The combined organic layers were washed withsaturated aqueous NaCl (300 mL), dried over MgSO₄, filtered, andconcentrated to give the crude product which was further purified bychromatography (hexanes:EtOAc=15:1) to yield the title compound (9.6 g)as a light yellow solid. LC-MS: 408 (M+Na).

A solution of Compound 4 (9.6 g, 25 mmol) in concentrated HCl (81 mL, 81mmol) was heated at 100° C. for 16 hours. The mixture was thenconcentrated to give the crude product which was further purified bywashing with Et₂O to yield Compound 5 (5.7 g) as a light yellow solidHCl salt. LC-MS: 320 (M+H).

To a solution of Compound 5 ((5.7 g, 18 mmol) in EtOH (10 mL) was added8M HCl in EtOH (120 mL, 960 mmol) at room temperature. The mixture washeated at 50° C. for 16 hours. After concentration, the crude productwas further purified by washing with Et₂O to yield the title compound(2.1 g) as a light yellow solid HCl salt. LC-MS: 348 (M+H).

Preparation 5:(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic Acid

1 M aqueous HCl (2.0 mmol) was added to(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (150.0 mg, 431 μmol) and the mixture was stirred at 100° C. for 2hours. The mixture was concentrated under vacuum for 3 hours and theresidue was purified by reverse phase preparative HPLC to yield thetitle compound (117 mg) as a white solid.

Preparation 6:(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid Ethyl Ester

t-Butanol (765 μL, 8.0 mmol) was added dropwise to a solution of oxalyldichloride (1.0 mL, 12.0 mmol) in DCM (5 mL) at 0° C. The mixture wasstirred at room temperature for 1 hour then concentrated in vacuo toyield chloro-oxo-acetic acid t-butyl ester as a clear colorless liquid(1.1 g). A 1 M solution in DCM was prepared by dissolving the liquid(1.1 g, 6.7 mmol) in DCM (6.7 mL).

DIPEA (3.0 mL, 17.2 mmol) was added to a solution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (2.0 g, 5.8 mmol) in DCM (20 mL) at 0° C. A 1 M solution ofchloro-oxo-acetic acid t-butyl ester in DCM (6.3 mL, 6.3 mmol) was addeddropwise and the resulting mixture was stirred at room temperature for15 minutes. The mixture was concentrated in vacuo to yield a clear brownliquid (1.4 g, 2.9 mmol). DCM (3.0 mL) and TFA (3.0 mL) were added andthe resulting mixture was stirred at room temperature for 1 hour. Themixture was then concentrated in vacuo to yield a brown liquid, whichwas purified (Interchim C18 column chromatography, 10 g column using40-95% MeCN in H₂O with 0.05% TFA) to yield the title compound as awhite solid (820 mg).

Preparation 7:(2R,4R)-4-Amino-5-(2′,5′-dichlorobiphenyl-4-yl)-2-hydroxypentanoic AcidEthyl Ester

To a solution of (S)-2-(4-bromobenzyl)-5-oxopyrrolidine-1-carboxylicacid t-butyl ester (33.5 g, 95 mmol) in 1,4-dioxane (1.2 L) was added2,5-dichlorophenylboronic acid (21.7 g, 114 mmol) and Pd(dppf)₂Cl₂ (3.5g, 4.7 mmol) at room temperature under nitrogen. After stirring for 10minutes, a solution of K₂CO₃ (26.1 g, 189 mmol) in water (120 mL) wasadded. The mixture was heated to 60° C. and stirred overnight. Afterevaporation of the solvent, water (400 mL) was added and extracted withEtOAc (3×400 mL). The combined organic layers were washed with saturatedaqueous NaCl (500 mL), dried over anhydrous Na₂SO₄, and concentrated toyield the crude product which was further purified by columnchromatography (hexanes:EtOAc=6:1) to yield Compound 1 (35.8 g) as alight yellow solid. LC-MS: 442 [M+Na].

To a solution of Compound 1 (35.8 g, 85 mmol) in anhydrous DCM (300 mL)was added TFA (30 mL, 405 mmol) at −5° C. under nitrogen. The mixturewas warmed to room temperature and stirred overnight. After evaporationof the solvent, the residue was diluted with EtOAc (500 mL), then washedwith saturated aqueous NaHCO₃ (3×300 mL), water (200 mL), and saturatedaqueous NaCl (250 mL), then dried over Na₂SO₄ and concentrated to yieldcrude Compound 2 (26 g) as a light yellow solid. LC-MS: 320 [M+H].

To a solution of Compound 2 (26 g, 81 mmol) in anhydrous THF (500 mL)was added dropwise n-butyllithium in hexane (39 mL, 97 mmol) over 1 hourat −78° C. under nitrogen. After stirring at −78° C. for 2 hours, thereaction was quenched by adding pivaloyl chloride (12.7 g, 105 mmol)dropwise over 30 minutes. After stirring at −78° C. for 2 hours, thereaction was quenched with saturated aqueous NH₄Cl (200 mL) andextracted with EtOAc (3×200 mL). The combined organic layers were washedwith saturated aqueous NaCl (300 mL), dried over anhydrous MgSO₄,filtered and concentrated to yield the crude product which was furtherpurified by chromatography (hexanes:EtOAc=25:1) to yield Compound 3 (33g) as a light yellow solid. LC-MS: 426 [M+Na].

To a solution of Compound 3 (10 g, 0.025 mol) in anhydrous THF (120 mL)was added dropwise NaHMDS (18.6 mL, 37 mmol) over 30 minutes at −78° C.under nitrogen. After stirring at −78° C. for 2 hours, a solution of(+)-(8,8-dichlorocamphorylsulfonyl)-oxaziridine (11.1 g, 37 mmol) in THF(80 mL) was added dropwise over 30 minutes. After stirring at −78° C.for 2 hours, the reaction was quenched with saturated aqueous NH₄Cl (500mL) and extracted with EtOAc (3×300 mL). The combined organic layerswere washed with saturated aqueous NaCl (300 mL), dried over MgSO₄,filtered and concentrated to yield the crude product which was furtherpurified by chromatography (hexanes:EtOAc=15:1) to yield Compound 4 (4.2g) as a light yellow oil. LC-MS: 442 [M+Na].

A solution of Compound 4 (4.2 g, 10 mmol) in concentrated HCl (80 mL,0.96 mol) was heated at 100° C. for 16 hours. The mixture was thenconcentrated to yield crude the product which was further purified bywashing with Et₂O to yield Compound 5 (3.8 g) as a white solid. LC-MS:354 [M+H].

To a solution of Compound 5 (3.8 g, 10 mmol) in EtOH (5 mL) was added 4MHCl in EtOH (100 mL, 0.4 mol) at room temperature. The mixture washeated at 50° C. for 16 hours. After concentration, the crude productwhich was further purified by washing with Et₂O to yield the titlecompound (3.3 g) as a white solid. LC-MS: 382 [M+H].

Preparation 8: (R)-4-Amino-5-biphenyl-4-yl-2-hydroxy-2-methyl-pentanoicAcid Ethyl Ester

To a solution of[(S)-1-biphenyl-4-ylmethyl-2-(2,2-dimethyl-4,6-dioxo-[1,3]dioxan-5-yl)-ethyl]-carbamicacid t-butyl ester (46 g, 0.1 mol) in t-butyl alcohol (100 mL) was addeddimethylmethyleneimmonium iodide (46.3 g, 0.3 mol) at room temperatureunder nitrogen. The mixture was heated to 65° C. and stirred at thistemperature for 16 hours. After filtration, the filtrate wasconcentrated to yield the crude product which was further purified bychromatography (hexanes:EtOAc=20:1˜10:1) to yield Compound 1 as a lightyellow solid) (18 g). LC-MS: [M+Na]: 460, [2M+Na]: 897.

To a solution of Compound 1 (18 g, 44 mmol) in acetone (430 mL) andwater (22 mL) was added Sudan Red as indicator. Ozone atmosphere wasintroduced into the mixture at 0° C. until the red color of Sudan Reddisappeared. Dimethyl sulfide (45 mL) was added and the mixture wasstirred at room temperature overnight. The mixture was then concentratedand the residue was purified by chromatography (hexanes:EtOAc=15:1˜7:1)to yield Compound 2 as a light yellow solid (9.5 g). LC-MS: [M+H]: 434,[2M+H]: 845.

To a solution of Compound 2 (9.5 g, 23 mmol) in anhydrous THF (120 mL)was added a solution of methylmagnesium bromide in THF (9.2 mL, 28 mmol)at −70° C. under nitrogen. The mixture was stirred at −60° C. for 3hours and the reaction was then quenched with saturated aqueous NH₄Cl(50 mL). The organic layer was separated and dried over MgSO₄. Themixture was then concentrated and the residue was purified bychromatography (hexanes:EtOAc=10:1˜5:1) to yield Compound 3 as an oil(7.9 g). LC-MS: [M+H]: 450, [2M+H]: 877.

To a solution of Compound 3 (7.9 g, 18.4 mmol) in anhydrous DCM (300 mL)was pumped HCl atmosphere at 0° C. for 6 hours. The mixture was thenconcentrated and the residue was washed with anhydrous Et₂O to yield thetitle compound as a white solid HCl salt (5.8 g). LC-MS: [M+H]: 364,[2M+H]: 727. ¹H NMR (300 MHz, DMSO): δ8.00-7.97 (d, 4H), 7.67-7.62 (m,6H), 7.47-7.28 (m, 8H), 6.32 (s, 1H), 6.09 (s, 1H), 4.13-4.06 (m, 2H),3.95-3.78 (m, 2H), 3.60 (s, 1H), 3.22-3.08 (m, 3H), 2.95-2.65 (m, 2H),1.99-1.79 (m, 4H), 1.30-0.87 (m, 9H).

Preparation 9:(3R,5R)-5-Amino-6-(4-bromo-2-chlorophenyl)-2-ethoxyhex-1-en-3-ol

To a suspension of 4-bromo-2-chlorobenzaldehyde (50 g, 22.8 mmol) inMeOH (500 mL) was added NaBH₄ (17.3 g, 45.6 mmol) in portions at 0° C.The mixture was stirred for 30 minutes and then aqueous NH₄Cl was addedto quench the reaction. The mixture was concentrated in vacuo. Theresidue was extracted with EtOAc (200 mL×2) and the combined organiclayers were dried over anhydrous Na₂SO₄, and concentrated under vacuumto yield Compound 1 (48 g) as a white solid.

To a solution of Compound 1 (46.8 g, 21.1 mmol) in dry DCM (500 mL) wasadded phosphorous tribromide (68.6 g, 25.3 mmol) dropwise at 0° C. undernitrogen. The mixture was stirred for 2 hours and then washed withsaturated aqueous NaHCO₃ (200 mL×2) and saturated aqueous NaCl (200 mL),dried over anhydrous Na₂SO₄, concentrated under vacuum to yield Compound2 (36 g) as a colorless oil.

To a stirred solution of (R)-pyrrolidine-2-carboxylic acid (57.7 g, 0.5mol) and KOH (84 g, 1.5 mol) in isopropyl alcohol (330 mL) was addedbenzyl chloride (70 mL, 0.6 mol) dropwise at 0° C. over 3 hours. Themixture was then stirred overnight at the same temperature. Theresulting mixture was neutralized with concentrate HCl to pH=6, followedby the addition of chloroform (200 mL). The mixture was stirred for 30minutes, then filtered and the precipitate was washed with chloroform(100 mL×3). The combined chloroform solutions were dried over anhydrousNa₂SO₄, and concentrated under vacuum to yield Compound 3 (52 g) as awhite solid. LC-MS: 206 [M+H]⁺.

To a solution of Compound 3 (10 g, 48.8 mmol) in dry DCM (50 mL) wasadded SO₂Cl₂ (7.3 g, 61 mmol) at −20° C. under nitrogen. The mixture wasstirred at −20° C. for 3 hours followed by the addition of a solution of(2-aminophenyl)(phenyl)methanone (6 g, 30.5 mmol) in dry DCM (25 mL) andthe mixture was stirred overnight at room temperature. A solution ofNa₂CO₃ (10.3 g) in water (40 mL) was added at 0° C. The organic layerwas separated and the aqueous layer was extracted with DCM (50 mL×3).The combined organic layers were dried over anhydrous Na₂SO₄ andconcentrated under vacuum. The residue was washed with MTBE (50 mL×2) toyield Compound 4 (8.5 g) as a yellow solid. LC-MS: 385 [M+H]⁺.

To a solution of Compound 4 (29.4 g, 76.5 mmol), glycine (28.7 g, 382.4mmol) and Ni(NO₃)₂.6H₂O (44.5 g, 152.9 mmol) in MeOH (280 mL) was addeda solution of KOH (30 g, 535.3 mmol) in MeOH (100 mL) at 45° C. undernitrogen. The mixture was stirred at 60° C. for an hour. The resultingsolution was neutralized with AcOH (31 mL) and poured into ice water(380 mL). The resulting solid was filtered and dissolved in DCM (450mL), which was washed with saturated aqueous NaCl (150 mL), dried overanhydrous Na₂SO₄ and concentrated. The residue was washed with EtOAc (50mL×2) to yield compound 5 (38 g) as a red solid. LC-MS: 498 [M+H]⁺.

Compound 5 (14.3 g, 28.7 mmol) and NaOH (3.4 g, 81.6 mmol) were added toa flask which was purged with nitrogen twice. Anhydrous DMF (100 mL) wasadded and the mixture was stirred for 5 minutes at 0° C. before asolution of Compound 2 (8.6 g, 30.1 mmol) in DMF (20 mL) was added. Thereaction mixture was stirred at room temperature for 30 minutes untilcomplete consumption of Compound 4 (checked by TLC). The resultingmixture was poured into a 5% AcOH aqueous solution (120 mL) which wasthen extracted with DCM (150 mL×3) and the combined organic layers werewashed with saturated aqueous NaCl (150 mL), dried over anhydrous Na₂SO₄and concentrated under vacuum. The residue was recrystallized withDCM/Et₂O (1:1) to yield Compound 6 (15.5 g) as a red solid. LC-MS: 702[M+H]⁺.

To a solution of Compound 6 (46 g, 65.6 mmol) in MeOH (300 mL) was added3N HCl (200 mL). The mixture was refluxed until the red color turnedgreen. The resulting solution was concentrated under vacuum andconcentrated NH₃.H₂O (100 mL) was added, and followed by the extractionwith DCM (200 mL×2). The aqueous phase was concentrated under vacuum andsubjected to the cation exchange resin (eluted with NH₃.H₂O/EtOH, 1:1)to yield Compound 7 (15 g) as a white solid. LC-MS: 280 [M+H]⁺.

To a suspension of Compound 7 (15 g, 53.9 mmol) in MeCN (150 mL) wasadded a solution of NaOH (4.3 g, 107.7 mmol) in water (150 mL) at 0° C.,and followed by the addition of (BOC)₂O (17.6 g, 80.8 mmol). The mixturewas stirred overnight at room temperature. The resulting solution wasconcentrated under vacuum, followed by the extraction with DCM (150mL×2). The aqueous phase was acidified with 1N HCl to pH=3 and extractedwith EtOAc (150 mL×3). The combined organic layers were washed withsaturated aqueous NaCl (150 mL), dried over anhydrous Na₂SO₄ andconcentrated under vacuum to yield Compound 8 (12.3 g, 60%) as a whitesolid. LC-MS: 402 [M+Na]⁺.

To a suspension of Compound 8 (18.4 g, 48.5 mmol) and Meldrum's acid(8.4 g, 58.2 mmol) in DCM (400 mL) was added DMAP (9.5 g, 77.6 mmol) at−5° C. After stirring for 10 minutes, a solution of DCC (12 g, 58.2mmol) in DCM (100 mL) was added dropwise at −5° C. The mixture wasstirred overnight at room temperature. The resulting solution was cooledto 0° C. and filtered. The filtrate was washed with aqueous citric acid(200 mL×3) and saturated aqueous NaCl (200 mL), dried over anhydrousNa₂SO₄, and concentrated under vacuum. The residue was washed with Et₂O(50 mL×2) to yield Compound 9 (22 g) as a light yellow solid.

To a solution of Compound 9 (22 g, 43.6 mmol) in DCM (400 mL) was addedAcOH (28.8 g, 479.4 mmol) at 0° C. After stirring for 10 minutes, NaBH₄(4.1 g, 109 mmol) was added in portions. The mixture was stirred for anhour at 0° C. The resulting solution was washed with saturated aqueousNaHCO₃ (200 mL×2) and saturated aqueous NaCl (200 mL), dried overanhydrous Na₂SO₄ and concentrated under vacuum. The residue was washedwith ether (100 mL×2) to yield Compound 10 (18.6 g) as an off-whitesolid. LC-MS: 514 [M+Na]⁺.

A solution of Compound 10 (18.6 g, 37.9 mmol) in toluene (350 mL) washeated under reflux for 2 hours. Upon cooling, the mixture wasevaporated to dryness to yield Compound 11 (14 g) as a yellow syrup.LC-MS: 334 [M-tBu+H]⁺.

To a solution of Compound 11 (14 g, 36.0 mmol) in DCM (250 mL) was addedTFA (20 mL). The mixture was stirred for 4 hours at 0° C. The resultingsolution was concentrated under vacuum to remove TFA. The residue wasdissolved in DCM (400 mL) and washed with saturated aqueous NaHCO₃ (200mL×2), dried over anhydrous Na₂SO₄ and concentrated to yield Compound 12(10 g) as a yellow solid. LC-MS: 290 [M+H]⁺.

To a solution of Compound 12 (10 g, 34.7 mmol) in dry THF (250 mL) wasadded NaH (2.4 g, 69.3 mmol, 70%) at 0° C. The mixture was stirred forone hour at 0° C. under nitrogen. Then pivaloyl chloride (5 g, 41.6mmol) was added. After stirring for another 2 hours, saturated aqueousNaHCO₃ (100 mL) was added to quench the reaction. The resulting mixturewas concentrated and extracted with EtOAc (100 mL×3) and the combinedorganic layers were washed with saturated aqueous NaCl (100 mL), driedover anhydrous Na₂SO₄ and concentrated under vacuum. The residue waspurified by silica gel chromatography (hexanes/EtOAc, 5:1) to yieldCompound 13 (11.8 g) as a white solid. LC-MS: 374[M+H]⁺.

To a solution of Compound 13 (11.8 g, 31.8 mmol) in dry THF (70 mL) wasadded NaHMDS (24 mL, 47.7 mmol, 2.0 M in THF) dropwise at −78° C. undernitrogen. After stirring for 30 minutes, a solution of(+)-(8,8-dichlorocamphorylsulfonyl)oxaziridine (15.2 g, 50.8 mmol) inTHF (70 mL) was added dropwise at −78° C. The mixture was stirred foranother hour at the same temperature before aqueous NH₄Cl (70 mL) wasadded to quench the reaction. The resulting mixture was extracted withEtOAc (150 mL×3) and the combined organic layers were washed withsaturated aqueous NaCl (150 mL), dried over anhydrous Na₂SO₄,concentrated under vacuum and purified by silica gel chromatography(hexanes/EtOAc, 20:1˜5:1) to yield the crude product (5 g), which wasfurther purified by preparative HPLC to yield Compound 14 (4 g) as ayellow solid. LC-MS: 390 [M+H]⁺.

A solution of Compound 14 (4 g, 10.3 mmol) in concentrated HCl (50 mL)was heated under reflux overnight. The mixture was concentrated undervacuum and the resulting solid was washed with Et₂O (50 mL×2) to yieldCompound 15 (3.1 g) as a white solid HCl salt. LC-MS: 324 [M+H]⁺.

A solution of Compound 15 (3.1 g, 8.6 mmol) in HCl/EtOH (6.7M, 40 mL)was stirred overnight at 50° C. The resulting mixture was concentratedunder vacuum and the residue was washed with ether (50 mL×2) to yieldthe title compound (2.9 g) as an off-white solid HCl salt. LC-MS: 352[M+H]⁺. ¹H NMR: (CD₃OD) 1.268 (t, J=6.9 Hz, 3H), 1.862-1.946 (m, 1H),2.068-2.143 (m, 1H), 3.104-3.199 (m, 2H), 3.769-3.809 (m, 1H),4.162-4.209 (m, 2H), 4.274-4.881 (m, 1H), 7.325 (dd, J=8.1, 2.1 Hz, 1H),7.522 (dd, J=8.3, 3.0 Hz, 1H), 7.696 (d, J=1.8 Hz, 1H).

Preparation 10:(2R,4S)-5-Biphenyl-4-yl-4-t-butoxycarbonylamino-2-hydroxymethylpentanoicAcid Ethyl Ester (compound 7) and(2S,4S)-5-Biphenyl-4-yl-4-t-butoxycarbonylamino-2-hydroxymethylpentanoicAcid Ethyl Ester (compound 8)

To a solution of (R)-3-biphenyl-4-yl-2-t-butoxycarbonylamino-propionicacid (50 g, 146 mmol), Meldrum's acid (23.3 g, 161 mmol) and DMAP (27.8g, 227 mmol) in anhydrous DCM (500 mL) was added a solution of DCC (33.3g, 161 mmol) in anhydrous DCM (200 mL) over 1 hour at −5° C. undernitrogen. The mixture was stirred at −5° C. for 8 hours, thenrefrigerated overnight, during which tiny crystals of dicyclohexylureaprecipitated. After filtration, the mixture was washed with 5% KHSO₄(4×200 mL) and saturated aqueous NaCl (1×200 mL), then dried underrefrigeration with MgSO₄ overnight. The solution was evaporated to yieldthe title compound (68 g, light yellow solid), which was used withoutfurther purification. LC-MS: 490 [M+Na], 957 [2M+Na].

To a solution of crude Compound 1 (68 g, 147 mmol) in anhydrous DCM (1L) was added AcOH (96.7 g, 1.6 mol) at −5° C. under nitrogen. Themixture was stirred at −5° C. for 0.5 hour, then NaBH₄ (13.9 g, 366mmol) was added in small portions over 1 hour. After stirring foranother 1 hour at −5° C., saturated aqueous NaCl (300 mL) was added. Theorganic layer was washed with saturated aqueous NaCl (2×300 mL) andwater (2×300 mL), dried over MgSO₄, filtered, and evaporated to yieldthe crude product, which was further purified by chromatography(hexanes:EtOAc=5:1) to yield Compound 2 (46 g, light yellow solid).LC-MS: 476 [M+Na], 929 [2M+Na].

A solution of Compound 2 (46 g, 101 mmol) in anhydrous toluene (300 mL)was refluxed under nitrogen for 3 hours. After evaporation of thesolvent, the residue was purified by chromatography (hexanes:EtOAc=10:1)to yield Compound 3 (27 g, light yellow solid). LC-MS: 374 [M+Na], 725[2M+Na]. ¹H NMR (300 MHz, CDCl3): δ7.64-7.62 (m, 4H), 7.51-7.46 (m, 2H),7.42-7.39 (m, 1H), 7.39-7.30 (m, 2H), 4.50-4.43 (m, 1H), 3.27-3.89 (m,1H), 2.88-2.80 (m, 1H), 2.48-2.42 (m, 2H), 2.09-1.88 (m, 2H), 1.66 (s,9H).

A mixture of Compound 3 (27 g, 77 mmol) andt-butoxy-N,N,N′,N′-tetramethylmethanediamine (40.3 g, 231 mmol) washeated to 80° C. under nitrogen. After stirring for 3 hours at 80° C.,the mixture was diluted with EtOAc (300 mL), washed with water (2×150mL) and saturated aqueous NaCl (2×150 mL), dried over MgSO₄, filtered,and evaporated to yield crude Compound 4 (29.7 g, light yellow oil).LC-MS: 425 [M+H], 835 [2M+H].

To a solution of crude Compound 4 (29.7 g, 73 mmol) in THF (200 mL) wasadded 1 M HCl (81 mL) at 0° C. under nitrogen. After stirring for 1 hourat room temperature, the mixture was diluted with EtOAc (100 mL) andadjusted with saturated aqueous NaHCO₃ to pH 7. The aqueous layer wasextracted with EtOAc (2×150 mL) and the combined organic layers werewashed with water (2×150 mL) and saturated aqueous NaCl(1×150 mL), driedover MgSO₄, filtered, and evaporated to yield crude Compound 5 (29.4 g,yellow oil). LC-MS: 402 [M+Na], 781 [2M+Na].

To a solution of Compound 5 (29.4 g, 77 mmol) in anhydrous THF (300 mL)was added anhydrous EtOH (30 mL) and AcOH (92.5 g, 1.5 mol) at −5° C.under nitrogen. The mixture was stirred at −5° C. for 0.5 hour, thenNaBH₃CN (19.4 g, 308 mmol) was added in small portions over 1 hour.After stirring for one additional hour at −5° C., the mixture wasadjusted with saturated aqueous NaHCO₃ to pH 7. The aqueous layers wereextracted with EtOAc (2×200 mL) and the combined organic layers werewashed with water (2×150 mL) and saturated aqueous NaCl (1×150 mL),dried over MgSO₄, filtered, and concentrated to yield the crude product,which was further purified by chromatography (hexanes:EtOAc=5:1) toyield Compound 6 (11.2 g, light yellow solid). LC-MS: 404 [M+Na], 785[2M+Na].

To a solution of Compound 6 (11.2 g, 29 mmol) in anhydrous EtOH (500 mL)was added anhydrous K₂CO₃ (8.0 g, 58 mmol) at 0° C. under nitrogen.After stirring for 1 hour at 0° C., the mixture was warmed to roomtemperature and stirred for 16 hours. After filtration, the filtrate wasconcentrated and the residual was diluted with water (150 mL), DCM (200mL) and saturated aqueous NaCl (50 mL). After separation, the aqueouslayer was extracted with DCM (2×150 mL). The combined organic layerswere washed with saturated aqueous NaCl (2×200 mL), dried over MgSO₄,and concentrated to yield the crude product which was further purifiedby column chromatography (hexanes:EtOAc=5:1) to yield Compounds 7 and 8(8.3 g, light yellow solid).

Compound 7: LC-MS: 450 [M+Na], 877 [2M+Na]. ¹H NMR (300 MHz, CDCl3):δ7.58-7.23 (m, 9H), 4.46-4.43 (d, 1H), 4.20-4.13 (m, 2H), 3.94 (s, 1H),3.82-3.70 (m, 2H), 2.85-2.70 (m, 3H), 2.25-2.22 (d, 1H), 2.01-1.92 (m,1H), 1.47 (s, 9H), 1.26-1.24 (m, 3H).

Compound 8: LC-MS: 450 [M+Na], 877 [2M+Na]. ¹H NMR (300 MHz, CDCl3):δ7.58-7.55 (m, 4H), 7.50-7.43 (m, 2H), 7.40-7.30 (m, 1H), 7.26-7.23 (m,1H), 4.46 (m, 1H), 4.21-4.13 (m, 2H), 3.94 (m, 1H), 3.82-3.77 (m, 2H),2.83-2.81 (d, 2H), 2.66-2.63 (m, 1H), 2.24 (m, 1H), 1.83-1.81 (m, 2H),1.38 (s, 9H), 1.30-1.25 (m, 3H).

Preparation 11:(2S,4S)-4-Amino-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethylpentanoic AcidEthyl Ester

To a solution of (S)-2-(4-bromobenzyl)-5-oxopyrrolidine-1-carboxylicacid t-butyl ester (18.4 g, 52 mmol) in 1,4-dioxane (500 mL) was added2-fluorophenylboronic acid (8.7 g, 63 mmol) and Pd(dppf)₂Cl₂ (3.8 g, 5.2mmol) at room temperature under nitrogen. After stirring for 10 minutes,a solution of K₂CO₃ (14.4 g, 104 mmol) in water (50 mL) was added. Themixture was heated to 80° C. and stirred at this temperature for 5hours. After evaporation of the solvent, water (300 mL) was added andthe mixture was extracted with EtOAc (3×200 mL). The combined organiclayers were washed with saturated aqueous NaCl (400 mL), dried overNa₂SO₄ and concentrated to give the crude product which was furtherpurified by column chromatography (hexanes:EtOAc=8:1) to yield Compound1 (17.3 g) as a red oil. LC-MS: 392 [M+Na].

A mixture of Compound 1 (17.3 g, 46.7 m mol) andt-butoxy-N,N,N′,N′-tetramethylmethanediamine (24.4 g, 140 mmol) washeated to 80° C. under nitrogen. After stirring for 3 hours at 80° C.,the mixture was diluted with EtOAc (300 mL) and washed with water (2×150mL), saturated aqueous NaCl (150 mL), dried over MgSO₄, filtered, andevaporated to yield crude Compound 2 (20.6 g) as a red oil. LC-MS: 425[M+H], 849 (2M+H).

To a solution of crude Compound 2 (20.6 g, 48.6 mmol) in THF (300 mL),was added 1 M HCl (58 mL, 58 mmol) at 0° C. under nitrogen. Afterstirring for 1 hour at room temperature, the mixture was diluted withEtOAc (100 mL) and adjusted with saturated aqueous NaHCO₃ to a pH of 7.The aqueous layer was extracted with EtOAc (2×150 mL) and the combinedorganic layers were washed with water (2×150 mL) and saturated aqueousNaCl (150 mL), dried over MgSO₄, filtered, and evaporated to yield thecrude Compound 3 (18.9 g) as a red oil. LC-MS: 420 (M+Na), 817 (2M+Na).

To a solution of crude Compound 3 (18.9 g, 47.6 mmol) in anhydrous THF(400 mL) was added anhydrous EtOH (50 mL) and AcOH (57.2 g, 952 mmol) at−5° C. under nitrogen. The mixture was stirred at −5° C. for 30 minutes,then NaBH₃CN (15 g, 238 mmol) was added in small portions over 1 hour.After stirring for an additional 1 hour at −5° C., the mixture wasadjusted with saturated aqueous NaHCO₃ to a pH of 7. The aqueous layerwas extracted with EtOAc (3×200 mL) and the combined organic layers werewashed with water (2×150 mL) and saturated aqueous NaCl (150 mL), driedover MgSO₄, filtered, and concentrated to yield the crude product whichwas further purified by chromatography (hexanes:EtOAc=6:1) to yieldCompound 4 (7.1 g) as a light yellow solid. LC-MS: 422 (M+Na), 821(2M+Na).

To a solution of Compound 4 (7.1 g, 17.7 mmol) in anhydrous EtOH (500mL) was added anhydrous K₂CO₃ (9.8 g, 70.8 mmol) at 0° C. undernitrogen. After stirring for 1 hour at 0° C., the mixture was warmed toroom temperature and stirred for 16 hours. After filtration, thefiltrate was concentrated and the residual was diluted with water (150mL), DCM (200 mL) and saturated aqueous NaCl (50 mL). After separation,the aqueous layer was extracted with DCM (2×150 mL). The combinedorganic layers were washed with saturated aqueous NaCl (2×200 mL), driedover MgSO₄, and concentrated to yield the crude product which wasfurther purified by column chromatography (hexanes:EtOAc=5:1) to yieldCompound 5 (2 g) as a light yellow solid. 2.1 g of the (R,S) isomer wasalso obtained as a light yellow solid.

Compound 5 (400 mg, 0.9 mmol) was dissolved in MeCN (3 mL) and 4 M HClin dioxane (0.5 mL). The mixture was stirred at room temperature for 1hour then concentrated to yield the title compound as an HCl salt (340mg), which was formed as an oil and solidified overnight.

Preparation 12:(2S,4R)-4-Amino-5-biphenyl-4-yl-2-hydroxymethyl-2-methylpentanoic AcidEthyl Ester

AcOH (8.6 mL) was added to a solution of crude[(R)-1-biphenyl-4-ylmethyl-2-(2,2-dimethyl-4,6-dioxo-[1,3]dioxan-5-yl)-2-oxo-ethyl]-carbamicacid t-butyl ester (6.4 g, 14 mmol) in anhydrous MeCN (90 mL) was addedAcOH (8.6 mL) at −5° C. under nitrogen. The mixture was stirred at −5°C. for 30 minutes, then sodium borohydride (1.3 g, 34.5 mmol) was addedin small portions over 2 hours. After stirring for another 1 hour at −5°C., saturated aqueous NaCl and 1.7 M of NaCl in water (30 mL) was added.The layers were separated and the organic layer was washed withsaturated aqueous NaCl (2×30 mL) and water (2×30 mL), dried under MgSO₄,filtered and evaporated, The resulting crude product was furtherpurified by chromatography (5:1 heptane:EtOAc) to yield Compound 1 (1.1g, purity 98.4%) as a light yellow solid.

Compound 1 (5.0 g, 11 mmol) and K₂CO₃ (1.8 g, 13.2 mmol) were stirred inDMF (33.9 mL) at 0° C. under nitrogen. Methyl iodide (892 μL) was addedand the resulting mixture was stirred at 0° C. for 1 hour. The mixturewas allowed to warm to room temperature. Saturated aqueous NaCl (35 mL)and EtOAc (35 mL) were added, and the resulting mixture was stirred for2 minutes. The organic layer was separated, dried (Na₂SO₄) and thesolvent evaporated. The residue was triturated with EtOAc (20 mL). Thesolid was filtered off and dried under vacuum. The filtrate wasconcentrated and triturated again with EtOAc to yield Compound 2 (3.9g).

Distilled water (140 mL) was purged 30 minutes under nitrogen, thencannulated into a vessel containing 0.1 M of samarium diiodide in THF(800 mL), exercising caution to not allow air into the reaction. Whilemaintaining an atmosphere of nitrogen, a degassed solution of Compound 2(3.7 g, 8.0 mmol) in THF (100 mL) was added via cannula. The resultingmixture was stirred for 15 minutes, then exposed to air. The solutionturned white. Saturated aqueous NaCl (12 mL), 10% citric acid (6 mL),and EtOAc (30 mL) were added. The mixture was stirred for 5 minutes,then the organic layer was separated, dried over Na₂SO₄ and concentratedin vacuo. The crude material was purified by normal phase chromatography(330 g gold column, 50% EtOAc with 0.5% AcOH/ether gradient). Thedesired diastereomer fractions were combined and the solvent wasconcentrated in vacuo. The BOC intermediate was stirred in MeCN (10 mL)and 4N HCl in dioxane (10 mL) for 30 min. The solvent was evaporated andthe product was azeotroped with toluene (2×) to yield Compound 3 (1.0 g)as an HCl salt.

Compound 3 (0.3 g, 957 μmol) was combined with EtOH (6 mL) and 4 M ofHCl in 1,4-dioxane (718 μL), and stirred overnight. The solvents wereevaporated and the product was azeotroped with toluene (2×) to yield thetitle compound (295 mg) as an HCl salt, which was used without furtherpurification.

Preparation 13:(2S,4R)-4-Amino-5-(3′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid

To a solution of (R)-2-amino-3-(4-bromophenyl)propionic acid (50 g, 0.2mol) in MeCN (700 mL) was added a solution of NaOH (16.4 g, 0.4 mol) inwater (700 mL) at −5° C. After stirring for 10 minutes, a solution of(BOC)₂O (44.7 g, 0.2 mol) in MeCN (100 mL) was added. The mixture waswarmed to room temperature and stirred overnight. After evaporation ofthe MeCN, the residue was diluted with DCM (800 mL) and acidified with 1M HCl to pH 2 at −5° C. The aqueous layer was extracted with DCM (3×200mL). The combined organic layers were washed with saturated aqueous NaCl(500 mL), dried over anhydrous Na₂SO₄ and concentrated to yield Compound1 (64.2 g, white solid). LC-MS: 366 [M+Na], 709 [2M+Na].

To a solution of Compound 1 (64.2 g, 187 mmol) in 1,4-dioxane (500 mL)was added 3-fluorophenylboronic acid (31.3 g, 224 mmol) and Pd(dppf)₂Cl₂(13.7 g, 19 mmol) at room temperature under nitrogen. After stirring for10 min, a solution of K₂CO₃ (51.7 g, 374 mmol) in water (250 mL) wasadded. The mixture was heated to 100° C. and stirred overnight. Afterevaporation of the solvent, water (200 mL) was added. The aqueous layerwas acidified with 1 M HCl to pH 2 and extracted with EtOAc (3×200 mL).The combined organic layers were washed with saturated aqueous NaCl (400mL), dried over anhydrous Na₂SO₄, and concentrated to yield the crudeproduct which was further purified by column chromatography(hexanes:EtOAc=4:1) to yield Compound 2 (45 g, light yellow oil). LC-MS:382 [M+Na], 741 [2M+Na].

To a solution of Compound 2 (45 g, 125 mmol), Meldrum's acid (23.5 g,163 mmol), and DMAP (26.0 g, 213 mmol) in anhydrous DCM (500 mL) wasadded a solution of DCC (33.3 g, 163 mmol) in anhydrous DCM (200 mL)over 1 hour at −5° C. under nitrogen. The mixture was stirred at −5° C.for 8 hours, then refrigerated overnight, during which tiny crystals ofdicyclohexylurea precipitated. After filtration, the mixture was washedwith 5% KHSO₄ (4×200 mL) and saturated aqueous NaCl (1×200 mL), thendried under refrigeration with anhydrous MgSO₄ overnight. The solutionwas evaporated to yield the crude Compound 3 (57.7 g, light yellow oil).LC-MS: 508 [M+Na], 993 [2M+Na].

To a solution of the crude Compound 3 (57.7 g, 119 mmol) in anhydrousDCM (1 L) was added AcOH (78.4 g, 1.3 mol) at −5° C. under nitrogen. Themixture was stirred at −5° C. for 0.5 hour, then NaBH₄ (11.3 g, 0.3 mol)was added in small portions over 1 hour. After stirring for another 1hour at −5° C., saturated aqueous NaCl (300 mL) was added. The organiclayer was washed with saturated aqueous NaCl (2×300 mL) and water (2×300mL), dried over anhydrous MgSO₄, filtered and concentrated to yield thecrude product, which was further purified by chromatography(hexanes:EtOAc=6:1) to yield Compound 4 (28 g, light yellow oil). LC-MS:494 [M+Na], 965 [2M+Na].

To a solution of Compound 4 (28 g, 60 mmol) in anhydrous DMF (250 mL)was added K₂CO₃ (9.9 g, 72 mmol) and CH₃I (25.6 g, 180 mmol) at 0° C.under nitrogen. After stirring for 1 hour at 0° C., the mixture waswarmed to room temperature and stirred overnight. The mixture wasdiluted with water (3 L) and extracted with EtOAc (3×300 mL). Thecombined organic layers were washed with saturated aqueous NaCl (500mL), dried over anhydrous Na₂SO₄, and concentrated to give the crudeproduct which was further purified by chromatography (hexanes:EtOAc=5:1)to yield Compound 5 (11.7 g, light yellow solid). LC-MS: 508 [M+Na], 993[2M+Na]. ¹H NMR (300 MHz, CD₃OD): δ7.52-7.49 (m, 2H), 7.41-7.39 (m, 2H),7.32-7.27 (m, 3H), 7.07-7.01 (m, 1H), 6.21-6.18 (d, 1H), 3.79 (m, 1H),2.78-2.61 (m, 2H), 2.35-2.20 (m, 2H), 1.76 (s, 6H), 1.59 (s, 3H), 2.21(s, 1H), 1.28 (s, 9H).

Distilled Water (181 mL) was purged 1 hour under nitrogen, thencannulated into a vessel containing 0.1 M of samarium diiodide in THF(800 mL). While maintaining an atmosphere of nitrogen, a similarlydegassed solution of Compound 5 (4.9 g, 10.0 mmol) and THF (20 mL) wasadded via cannula. The resulting mixture was stirred for 15 minutes,then exposed to air. A white solid formed. The THF was evaporated, andEtOAc (200 mL) was added followed by saturated aqueous NaCl (50 mL) and10% citric acid (20 mL). The mixture was stirred for 5 minutes, then theorganic layer was separated, dried over Na₂SO₄ and concentrated invacuo. The crude product was purified by normal phase chromatography(330 g gold column, 1:1 ether:EtOAc with 0.5% AcOH). The fractioncontaining the desired diastereomers were combined. The solvent wasevaporated in vacuo to yield the BOC-protected acid (1.5 g). The BOC wascleaved by stirring the intermediate in 4M HCl in dioxane (6 mL) andMeCN (10 mL) for 30 minutes. The solvent was then evaporated in vacuo toyield the title compound as an HCl salt.

Preparation 14:[(R)-1-(4-Bromobenzyl)-2-(2,2,5-trimethyl-4,6-dioxo-[1,3]dioxan-5-yl)ethyl]carbamicAcid t-Butyl Ester

To a mixture of (R)-2-amino-3-(4-bromophenyl)propionic acid (100 g, 410μmol) in MeCN (600 mL) was added dropwise a solution of NaOH (32.8 g,820 μmol) in water (800 mL) at 0° C. The resulting solution was stirredfor 30 minutes. A solution of (BOC)₂O (93.8 g, 430 μmol) in MeCN (200mL) was added, and the resulting mixture was warmed to room temperatureand stirred overnight. The MeCN was evaporated and the residue wasdiluted with DCM (1 L) and acidified with 2 M HCl to pH=2 at −5° C. Theaqueous layer was extracted with DCM and the combined organic layerswere washed with saturated aqueous NaCl (500 mL), dried over anhydrousNa₂SO₄ and concentrated to yield crude Compound 1 (141 g, 100%) as ayellow solid. LC-MS: 366[M+Na]⁺.

Compound 1 (20 g, 58.1 mmol) was combined with2,2-dimethyl-1,3-dioxane-4,6-dione (9.2 g, 63.9 mmol), DMAP (10.7 g,87.2 mmol), and anhydrous DCM (400 mL), and cooled to 0° C. Afterstirring for 30 minutes, a solution of DCC (13.2 g, 63.9 mmol) in DCM(50 mL) was added dropwise at 0° C. under nitrogen. After the addition,the ice bath was removed and the mixture was stirred at room temperatureovernight. The solution was cooled at −20° C. for 1 hour and then thesolids were filtered off. The filtrate was washed with a 5% KHSO₄solution (4×100 mL) and saturated aqueous NaCl (200 mL). The organiclayer was dried over anhydrous Na₂SO₄ and evaporated to yield crudeCompound 2 (27.5 g) as a gray solid. LC-MS: 492 [M+Na]⁺.

To a solution of Compound 2 (27.5 g, 58.1 mmol) in anhydrous DCM (400mL) was added AcOH (38.4 g, 639.1 mmol) at −5° C. under nitrogen. Themixture was stirred at −5° C. for 30 minutes. NaBH₄ (5.5, 145.2 mmol)was added in portions over 30 minutes, and the resulting solution wasstirred at room temperature for 3 hours. Saturated aqueous NaCl (300 mL)was added to quench the reaction. The organic layer was washed withsaturated aqueous NaCl (2×200 mL), dried over anhydrous Na₂SO₄ andconcentrated to yield crude Compound 3 (22.6 g). LC-MS: 478 [M+Na]⁺.

To a solution of Compound 3 (22.6 g, 49.6 mmol) and K₂CO₃ (8.3 g, 59.5mmol) in anhydrous DMF (160 mL) was added methyl iodide (14 g, 99.2mmol) dropwise at 0° C. After the addition, the solution was stirred atroom temperature overnight. The mixture was evaporated and the residuewas dissolved in EtOAc (500 mL) and washed with saturated aqueous NaCl(2×200 mL). The organic solution was dried over anhydrous Na₂SO₄ andconcentrated to yield the crude product which was triturated with ethylether (100 mL), then filtered to yield the title compound (14.5 g) as awhite solid. LC-MS: 492 [M+Na]⁺.

Preparation 15:(2S,4R)-4-t-Butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid

A mixture of[(R)-1-(4-bromobenzyl)-2-(2,2,5-trimethyl-4,6-dioxo-[1,3]dioxan-5-yl)ethyl]carbamicacid t-butyl ester (8 g, 17 mmol), 3-chlorophenylboronic acid (3 g, 18.7mmol), Pd(dppf)₂Cl₂ (400 mg, 550 μmol) and potassium fluoride (2 g, 34mmol) in water (80 mL) and dioxane (80 mL) was stirred at 60° C. underargon for 3 hours. The mixture was concentrated, dispersed in water (150mL), extracted with EtOAc (2×100 mL), dried over anhydrous Na₂SO₄ andevaporated to yield the crude product, which was purified by columnchromatography (PE:EtOAc=10:1) to yield Compound 1 (7 g) as a whitesolid. LC-MS: 524 [M+Na]⁺

Samarium powder (50 g, 330 μmol) was flushed with argon (20 minutes).Anhydrous THF (1.5 L) was added and the resulting suspension was bubbledwith argon (15 minutes). Iodine (70 g, 270 mmol) was added and themixture was flushed again with argon (10 minutes). The mixture wascovered with aluminum foil and heated at 65° C. overnight then allowedto cool to room temperature. A solution of Compound 1 (7 g, 13.9 mmol)in THF (200 mL) and water (100 mL) was sealed and flushed with argon (10minutes), cooled to −70° C., flushed with argon (10 minutes), cooled to−70° C., and flushed with argon (30 minutes). The samarium powdersolution (1.5 L) was then added to the cooled solution via cannula, andstirred at room temperature for 2 hours. The solution was evaporated,and the residue was dissolved in EtOAc (200 mL), washed with tartaricacid solution (10%, 150 mL), dried over anhydrous Na₂SO₄, concentratedand purified by column chromatography (PE:EtOAc=0 to 30%, added with0.05% AcOH) to yield the title compound (3 g) as a white solid. LC-MS:470 [M+Na]⁺. ¹H NMR (300 MHz, CD3OD): δ7.28˜7.56 (m, 8H), 3.94 (s, 1H),3.56˜3.66 (m, 2H), 2.69˜2.82 (m, 2H), 1.70˜1.90 (m, 2H), 1.17˜1.31 (m,12H).

Preparation 16:(2S,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid

(2S,4R)-4-t-Butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (773 mg, 1.8 mmol) was combined with MeCN (4 mL) and 4N HCl indioxane (1 mL) and stirred for 20 minutes. The reaction mixture wasconcentrated under reduced pressure then purified (Interchimreverse-phase) to yield the title compound as a TFA salt.

Example 1(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(2-oxobutyrylamino)pentanoicAcid

(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (50 mg, 144 μmol) was added to a solution of 2-oxo-butyric acid(1.2 eq., 165 μmol) and HATU (71.1 mg, 187 μmol) in DMF (250 μL),followed by the addition of DIPEA (75 μL, 431 μmol). The resultingmixture was stirred at room temperature for 1 hour. 10 M aqueous LiOH(115 μL, 1.2 mmol) was added and the mixture was stirred for anadditional 1 hour. The mixture was washed with DCM (1 mL) and theaqueous layer was acidified with concentrated HCl (250 μL) and extractedwith DCM (2×1 mL). The DCM extracts were combined and concentrated invacuo, and the resulting crude liquid was purified by reverse phasepreparative HPLC to yield the title compound (2.9 mg). MS m/z[M+H]⁺calc'd for C₂₁H₂₂ClNO₅, 404.12. found 404.2.

Example 2

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, thefollowing compounds were prepared as the parent compound. Thesecompounds, where X is —C(O)—R⁶, are depicted by formula IIa:

R¹ and R⁴═H; R⁵═Cl

MS m/z: [M + H]⁺ Ex. R⁶ Formula calcd found 1 —CH₂CH—(CH₃)₂ C₂₃H₂₆ClNO₅432.15 432.2 2 benzyl C₂₆H₂₄ClNO₅ 466.13 466.2

-   1.    (2R,4R)-5-(3′-Chloro-biphenyl-4-yl)-2-hydroxy-4-(4-methyl-2-oxo-pentanoylamino)-pentanoic    acid-   2.    (2R,4R)-5-(3′-Chloro-biphenyl-4-yl)-2-hydroxy-4-(2-oxo-3-phenyl-propionylamino)-pentanoic    acid

Example 3(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-((S)-2-hydroxymethylpyrrolidin-1-yl)-2-oxo-acetylamino]pentanoicAcid

DIPEA (62 μl, 357 μmol) was added to a solution of(S)-1-pyrrolidin-2-yl-methanol (L-prolinol; 18 μL, 179 μmol),(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicacid ethyl ester (50 mg, 119 μmol) and HATU (58.9 mg, 155 μmol) in DMF(0.5 mL), and the resulting mixture was stirred at room temperature for10 minutes. 5 M aqueous LiOH (191 μL, 953 μmol) was added and themixture was stirred at room temperature for 10 minutes. AcOH (2 mL) wasadded and the mixture was purified (Interchim reverse-phase 30 g columnusing H₂O/MeCN 30-95%) to yield the title compound as a white solid(29.5 mg). MS m/z [M+H]⁺calc'd for C₂₄H₂₇C1N₂O₆, 475.16. found 475.2.

Example 4

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, thefollowing compounds were prepared as the parent compound or as a TFAsalt. These compounds, where X is —C(O)—NR⁷R⁸, are depicted by formulaIIb:

R¹ and R⁴═H; R⁵═Cl

MS m/z: [M + H]⁺ Ex. R⁷ R⁸ Formula calcd found  1 H cyclopentylC₂₄H₂₇ClN₂O₅ 459.16 459.2  2 H

C₂₄H₂₁BrClN₃O₅ 546.04 546.2  3 H

C₂₄H₂₂ClN₃O₅ 468.12 468.2  4 H

C₂₅H₂₄ClN₃O₅ 482.14 482.2  5 H —O-benzyl C₂₆H₂₅ClN₂O₆ 497.14 497.2  6 H—O—CH₃ C₂₀H₂₁ClN₂O₆ 421.11 421.2  7 H

C₂₂H₂₂ClN₃O₇ 476.11 476.2  8 H

C₂₃H₂₆ClN₃O₆ 476.15 476.2  9 OH phenyl C₂₅H₂₃ClN₂O₆ 483.12 483.2 10 OH—CH₃ C₂₀H₂₁ClN₂O₆ 421.11 421.2 11 —CH₃ —CH₃ C₂₁H₂₃ClN₂O₅ 419.13 419.2 12—CH₃ —O—CH₃ C₂₁H₂₃ClN₂O₆ 435.12 435.1

-   1.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-(cyclopentylaminooxalylamino)-2-hydroxypentanoic    acid-   2.    (2R,4R)-4-[(6-Bromopyridin-3-ylaminooxalyl)amino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic    acid-   3.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(pyridin-4-ylaminooxalyl)-amino]-pentanoic    acid-   4.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(4-methylpyridin-3-ylaminooxalyl)amino]pentanoic    acid-   5.    (2R,4R)-4-(Benzyloxyaminooxalylamino)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-pentanoic    acid-   6.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(methoxyaminooxalylamino)-pentanoic    acid-   7.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3-oxo-isoxazolidin-4-ylaminooxalyl)-amino]pentanoic    acid-   8.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(morpholin-4-ylaminooxalyl)-amino]pentanoic    acid-   9.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(hydroxyphenylaminooxalyl)-amino]pentanoic    acid-   10.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(hydroxymethylaminooxalyl)-amino]pentanoic    acid-   11.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-(dimethylaminooxalylamino)-2-hydroxy-pentanoic    acid-   12.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(methoxymethylaminooxalyl)-amino]pentanoic    acid

MS m/z: [M + H]⁺ Ex. R⁷ and R⁸ taken together to form a ring Formulacalcd found 13

C₂₂H₂₃ClN₂O₆ 447.12 447.2 14

C₂₂H₂₁ClF₂N₂O₅ 467.11 467.2 15

C₂₄H₂₇ClN₂O₇S 523.12 523.2 16

C₂₄H₂₇ClN₂O₆ 475.16 475.2 17

C₂₄H₂₆ClN₃O₆ 488.15 488.2 18

C₂₄H₂₇ClN₂O₆ 475.16 475.2 19

C₂₆H₃₁ClN₂O₆ 503.19 503.2 20

C₂₅H₂₉ClN₂O₆ 489.17 489.2 21

C₂₄H₂₇ClN₂O₆ 475.16 475.2 22

C₂₅H₂₉ClN₂O₆ 489.17 489.2 23

C₂₉H₃₀ClN₃O₅ 536.19 536.2 24

C₂₆H₃₁ClN₂O₆ 503.19 503.2 25

C₂₅H₂₉ClN₂O₆ 489.17 489.2 26

C₂₅H₂₉ClN₂O₆ 489.17 489.2 27

C₂₅H₂₇ClN₂O₇ 503.15 503.2 28

C₂₅H₂₇ClN₂O₇ 503.15 503.2 29

C₂₅H₂₈ClN₃O₆ 502.17 502.2 30

C₂₅H₂₆ClN₃O₅ 484.16 484.2 31

C₂₄H₂₆ClFN₂O₅ 477.15 477.2 32

C₂₆H₃₁ClN₂O₆ 503.19 503.2 33

C₂₅H₂₉ClN₂O₆ 489.17 489.2 34

C₂₄H₂₈ClN₃O₅ 474.17 474.2 35

C₂₆H₃₀ClN₃O₇ 532.18 532.2 36

C₂₈H₂₈ClN₃O₇ 554.16 554.4 37

C₃₀H₂₉ClN₄O₅ 561.18 561.2 38

C₂₇H₃₄ClN₃O₆ 532.21 532.2 39

C₂₄H₂₈ClN₃O₇S 538.13 538.2 40

C₂₆H₃₁ClN₄O₆ 531.19 531.2 41

C₂₅H₂₈ClN₃O₇ 518.16 518.2 42

C₂₅H₂₉ClN₂O₆ 489.17 489.2 43

C₂₄H₂₇ClN₂O₇ 491.15 491.2 44

C₂₃H₂₅ClN₂O₆ 461.14 461.2 45

C₂₅H₂₉ClN₂O₆ 489.17 489.2 46

C₂₅H₂₉ClN₂O₆ 489.17 489.2 47

C₂₅H₂₉ClN₂O₆ 489.17 489.2 48

C₂₂H₂₃ClN₂O₆ 447.12 447.0 49

C₂₂H₂₃ClN₂O₇ 463.12 463.0 50

C₂₄H₂₅ClN₂O₆ 473.14 473.2

-   13.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(3-hydroxyazetidin-1-yl)-2-oxo-acetylamino]pentanoic    acid-   14.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-[2-(3,3-difluoroazetidin-1-yl)-2-oxo-acetylamino]-2-hydroxypentanoic    acid-   15.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(3-methanesulfonyl-pyrrolidin-1-yl)-2-oxo-acetylamino]pentanoic    acid-   16.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-((R)-2-hydroxymethylpyrrolidin-1-yl)-2-oxo-acetylamino]pentanoic    acid-   17.    (2R,4R)-4-[2-(3-Carbamoylpyrrolidin-1-yl)-2-oxo-acetylamino]-5-(3′-chloro-biphenyl-4-yl)-2-hydroxypentanoic    acid-   18.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(4-hydroxy-piperidin-1-yl)-2-oxo-acetylamino]pentanoic    acid-   19.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(4-methoxymethyl-piperidin-1-yl)-2-oxo-acetylamino]pentanoic    acid-   20.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(4-hydroxymethyl-piperidin-1-yl)-2-oxo-acetylamino]pentanoic    acid-   21.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-((R)-3-hydroxy-piperidin-1-yl)-2-oxo-acetylamino]pentanoic    acid-   22.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(4-hydroxy-4-methyl-piperidin-1-yl)-2-oxo-acetylamino]pentanoic    acid-   23.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-oxo-2-(3,4,5,6-tetrahydro-2H-[4,4]bipyridinyl-1-yl)-acetylamino]pentanoic    acid-   24.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-{2-[4-(2-hydroxyethyl)-piperidin-1-yl]-2-oxo-acetylamino}pentanoic    acid-   25.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-((R)-2-hydroxymethyl-piperidin-1-yl)-2-oxo-acetylamino]pentanoic    acid-   26.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-((S)-2-hydroxymethyl-piperidin-1-yl)-2-oxo-acetylamino]pentanoic    acid-   27.    1-[(1R,3R)-3-Carboxy-1-(3′-chlorobiphenyl-4-ylmethyl)-3-hydroxypropyl-aminooxalyl]-piperidine-4-carboxylic    acid-   28.    1-[(1R,3R)-3-Carboxy-1-(3′-chlorobiphenyl-4-ylmethyl)-3-hydroxypropyl-aminooxalyl]-piperidine-3-carboxylic    acid-   29.    (2R,4R)-4-[2-(4-Carbamoyl-piperidin-1-yl)-2-oxo-acetylamino]-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic    acid-   30.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-[2-(3-cyanopiperidin-1-yl)-2-oxo-acetylamino]-2-hydroxypentanoic    acid-   31.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-[2-(4-fluoropiperidin-1-yl)-2-oxo-acetylamino]-2-hydroxypentanoic    acid-   32.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-{2-[3-(2-hydroxyethyl)-piperidin-1-yl]-2-oxo-acetylamino}pentanoic    acid-   33.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-((S)-3-hydroxymethyl-piperidin-1-yl)-2-oxo-acetylamino]pentanoic    acid-   34.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(4-methyl-piperazin-1-yl)-2-oxo-acetylamino]pentanoic    acid-   35.    4-[(1R,3R)-3-Carboxy-1-(3′-chlorobiphenyl-4-ylmethyl)-3-hydroxy-propylaminooxalyl]-piperazine-1-carboxylic    acid ethyl ester-   36.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{2-[4-(furan-2-carbonyl)-piperazin-1-yl]-2-oxo-acetylamino}-2-hydroxypentanoic    acid-   37.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{2-[4-(2-cyano-phenyl)-piperazin-1-yl]-2-oxo-acetylamino}-2-hydroxypentanoic    acid-   38.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{2-[4-(2-ethoxy-ethyl)-piperazin-1-yl]-2-oxo-acetylamino}-2-hydroxypentanoic    acid-   39.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(4-methanesulfonyl-piperazin-1-yl)-2-oxo-acetylamino]pentanoic    acid-   40.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-[2-(4-dimethylcarbamoyl-piperazin-1-yl)-2-oxo-acetylamino]-2-hydroxypentanoic    acid-   41.    4-[(1R,3R)-3-Carboxy-1-(3′-chlorobiphenyl-4-ylmethyl)-3-hydroxy-propylaminooxalyl]-piperazine-1-carboxylic    acid methyl ester-   42.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-[2-((3R,5S)-3,5-dimethylmorpholin-4-yl)-2-oxo-acetylamino]-2-hydroxypentanoic    acid-   43.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(2-hydroxymethyl-morpholin-4-yl)-2-oxo-acetylamino]pentanoic    acid-   44.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(2-morpholin-4-yl-2-oxo-acetylamino)pentanoic    acid-   45.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-[2-((2R,6S)-2,6-dimethylmorpholin-4-yl)-2-oxo-acetylamino]-2-hydroxypentanoic    acid-   46.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-[2-((2S,5R)-2,5-dimethylmorpholin-4-yl)-2-oxo-acetylamino]-2-hydroxypentanoic    acid-   47.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-[2-((3S,5S)-3,5-dimethylmorpholin-4-yl)-2-oxo-acetylamino]-2-hydroxypentanoic    acid-   48.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(2-isoxazolidin-2-yl-2-oxo-acetylamino)pentanoic    acid-   49.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(4-hydroxyisoxazolidin-2-yl)-2-oxo-acetylamino]pentanoic    acid-   50.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(2-oxa-6-aza-spiro[3.3]hept-6-yl)-2-oxo-acetylamino]pentanoic    acid

Example 5(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{[N′-(2,4-dinitrophenyl)-hydrazinooxalyl]-amino}-2-hydroxypentanoicAcid

DIPEA (31 μl, 179 μmol) was added to a solution of(2,4-dinitrophenyl)-hydrazine (14 mg, 71 μmol),(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicacid ethyl ester (25 mg, 60 μmol) and HATU (34 mg, 89 μmol) in DMF (0.3mL), and the resulting mixture was stirred at room temperature for 15minutes. 5 M aqueous LiOH (95 μL, 476 μmol) was added and the mixturewas stirred at room temperature for 15 minutes. AcOH (1 mL) was addedand the mixture was purified by purified by reverse phase preparativeHPLC to yield the title compound as a TFA salt (7.8 mg). MS m/z[M+H]⁺calc'd for C₂₅H₂₂C1N₅O₉, 572.11. found 572.2.

Example 6

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, thefollowing compounds were prepared as the parent compound or as a TFAsalt. These compounds, where X is —C(O)—NR⁹—NR¹⁰R¹¹, are depicted byformula IIc:

R¹, R⁴, R⁹, and R¹⁰═H; R⁵═Cl

MS m/z: [M + H]⁺ Ex. R¹¹ Formula calcd found  1 —CH₃ C₂₀H₂₂ClN₃O₅ 420.12419.2  2 —CH₂CH(CH₃)₂ C₂₃H₂₈ClN₃O₅ 462.17 462.2  3

C₂₄H₂₃ClN₄O₅ 483.14 483.2  4

C₂₄H₂₃ClN₄O₅ 483.14 483.2  5

C₂₃H₂₂ClN₅O₆ 500.13 500.2  6

C₂₂H₂₄ClN₅O₅ 474.15 474.2  7

C₂₅H₂₃Cl₂N₃O₅ 516.10 516.2  8

C₂₅H₂₃ClFN₃O₅ 500.13 500.2  9

C₂₅H₂₃Cl₂N₃O₅ 516.10 516.2 10

C₂₆H₂₆ClN₃O₅ 496.16 496.2 11

C₂₆H₂₆ClN₃O₅ 496.16 496.2 12

C₂₅H₂₃ClFN₃O₅ 500.13 500.2 13

C₂₅H₂₃Cl₂N₃O₅ 516.10 516.2 14

C₂₅H₂₃BrClN₃O₅ 560.05 560.2 15

C₂₆H₂₆ClN₃O₆ 512.15 512.2 16

C₂₆H₂₆ClN₃O₅ 496.16 496.2 17

C₂₅H₂₄ClN₃O₅ 482.14 482.2

-   1.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(N′-methylhydrazinooxalyl)-amino]pentanoic    acid-   2.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(N′-isobutylhydrazinooxalyl)-amino]pentanoic    acid-   3.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(N′-pyridin-4-yl-hydrazinooxalyl)-amino]pentanoic    acid-   4.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(N′-pyridin-3-yl-hydrazinooxalyl)-amino]pentanoic    acid-   5.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-{[N′-(6-hydroxy-pyrimidin-4-yl)-hydrazinooxalyl]amino}-pentanoic    acid-   6.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{[N′-(4,5-dihydro-1H-imidazol-2-yl)-hydrazinooxalyl]-amino}-2-hydroxypentanoic    acid-   7.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{[N′-(2-chloro-phenyl)-hydrazinooxalyl]-amino}-2-hydroxypentanoic    acid-   8.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{[N′-(2-fluoro-phenyl)-hydrazinooxalyl]-amino}-2-hydroxypentanoic    acid-   9.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{[N′-(3-chloro-phenyl)-hydrazinooxalyl]-amino}-2-hydroxypentanoic    acid-   10.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(N′-o-tolyl-hydrazinooxalyl)-amino]-pentanoic    acid-   11.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(N′-p-tolyl-hydrazinooxalyl)-amino]-pentanoic    acid-   12.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{[N′-(4-fluoro-phenyl)-hydrazinooxalyl]-amino}-2-hydroxypentanoic    acid-   13.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{[N′-(4-chloro-phenyl)-hydrazinooxalyl]-amino}-2-hydroxypentanoic    acid-   14.    (2R,4R)-4-{[N′-(4-Bromophenyl)-hydrazinooxalyl]-amino}-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic    acid-   15.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-{[N′-(4-methoxyphenyl)-hydrazinooxalyl]amino}pentanoic    acid-   16.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(N′-m-tolylhydrazinooxalyl)-amino]pentanoic    acid-   17.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(N′-phenylhydrazinooxalyl)-amino]pentanoic    acid

Example 7(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-{2-[N′-(2-hydroxybenzoyl)hydrazino]-2-oxo-acetylamino}pentanoicAcid

DIPEA (31 μl, 179 μmol) was added to a solution of(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicacid ethyl ester (25 mg, 60 μmol), HATU (34 mg, 89 μmol), and2-hydroxybenzoic acid hydrazide (11 mg, 71 μmol), in DMF (0.3 mL), andthe resulting mixture was stirred at room temperature for 15 minutes. 5M aqueous LiOH (95 μL, 476 μmol) was added and the mixture was stirredat room temperature for 15 minutes. AcOH (1 mL) was added and themixture was purified by purified by reverse phase preparative HPLC toyield the title compound (0.9 mg). MS m/z [M+H]⁺calc'd for C₂₆H₂₄C1N₃O₇,526.13. found 526.2.

Example 8

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, thefollowing compounds were prepared as the parent compound or as a TFAsalt. These compounds, where X is —C(O)—NR¹²—NR¹³—C(O)—R¹⁴, are depictedby formula IId:

R¹, R⁴, R¹², and R¹³═H; R⁵═Cl

MS m/z: [M + H]⁺ Ex. R¹⁴ Formula calcd found 1

C₂₇H₂₆ClN₃O₇ 540.15 540.2 2

C₂₅H₂₂Cl₂N₄O₆ 545.09 545.2 3

C₂₅H₂₃ClN₄O₆ 511.13 511.2 4

C₂₅H₂₃ClN₄O₆ 511.13 511.2 5

C₂₄H₂₂ClN₃O₇ 500.11 500.2 6

C₂₆H₂₃ClN₄O₈ 555.12 555.9

-   1.    (2R,4R)-4-[2-(N′-Benzyloxycarbonyl-hydrazino)-2-oxo-acetylamino]-5-(3′-chloro-biphenyl-4-yl)-2-hydroxypentanoic    acid-   2.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{2-[N′-(6-chloro-pyridine-3-carbonyl)-hydrazino]-2-oxo-acetylamino}-2-hydroxypentanoic    acid-   3.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-{2-oxo-2-[N′-(pyridine-3-carbonyl)-hydrazino]acetylamino}pentanoic    acid-   4.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-{2-oxo-2-[N′-(pyridine-4-carbonyl)-hydrazino]acetylamino}pentanoic    acid-   5.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-{2-[N′-(furan-2-carbonyl)-hydrazino]-2-oxo-acetylamino}-2-hydroxypentanoic    acid-   6.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-{2-[N′-(2-nitro-benzoyl)-hydrazino]-2-oxo-acetylamino}pentanoic    acid

Example 9(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(tetrahydropyran-2-carbonyl)amino]pentanoicAcid

(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (50 mg, 144 μmol) was added to a solution oftetrahydro-pyran-2-carboxylic acid (1.2 eq., 165 μmol) and HATU (71.1mg, 187 μmol) in DMF (250 μL), followed by the addition of DIPEA (75 μL,431 μmol). The resulting mixture was stirred at room temperature for 1hour. 10 M aqueous LiOH (115 μL, 1.2 mmol) was added and the mixture wasstirred for an additional 1 hour. The mixture was washed with DCM (1 mL)and the aqueous layer was acidified with concentrated HCl (250 μL) andextracted with DCM (2×1 mL). The DCM extracts were combined andconcentrated in vacuo, and the resulting crude liquid was purified byreverse phase preparative HPLC to yield the title compound (32.6 mg). MSm/z [M+H]⁺calc'd for C₂₃H₂₆ClNO₅, 432.15. found 432.2.

Example 10

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, thefollowing compounds were prepared as the parent compound. Thesecompounds, where X is —CH(R¹⁵)—OR¹⁶, are depicted by formula He:

R¹, R⁴, and R¹⁵═H; R⁵═Cl

MS m/z: [M + H]⁺ Ex. R¹⁶ Formula calcd found 1 H C₁₉H₂₀ClNO₅ 378.10378.2 2 —CH₃ C₂₀H₂₂ClNO₅ 392.12 392.2 3 —CH₂CH₃ C₂₁H₂₄ClNO₅ 406.13 406.24 —CH(CH₃)₂ C₂₂H₂₆ClNO₅ 420.15 420.2 5

C₂₄H₂₃ClN₂O₅ 455.13 455.2 6 benzyl C₂₆H₂₆ClNO₅ 468.15 468.2 7 phenylC₂₅H₂₄ClNO₅ 454.13 454.2 8

C₂₅H₂₄ClNO₆ 470.13 470.2 9

C₂₆H₂₆ClNO₆ 484.14 484.2

-   1.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(2-hydroxyacetylamino)-pentanoic    acid-   2.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(2-methoxyacetylamino)-pentanoic    acid-   3.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-(2-ethoxyacetylamino)-2-hydroxy-pentanoic    acid-   4.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(2-isopropoxyacetylamino)-pentanoic    acid-   5.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(pyridin-3-yloxy)-acetylamino]-pentanoic    acid-   6.    (2R,4R)-4-(2-Benzyloxy-acetylamino)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-pentanoic    acid-   7.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(2-phenoxyacetylamino)-pentanoic    acid-   8.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(4-hydroxyphenoxy)-acetylamino]-pentanoic    acid-   9.    (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[2-(3-methoxyphenoxy)-acetylamino]-pentanoic    acid

Example 11(2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxymethyl-4-(2-methoxyacetylamino)-2-methylpentanoicAcid

Methoxy-acetyl chloride (7 mg, 30 μmol) was combined with a 0.4 mLsolution of 0.075 M of(2S,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-pentanoicacid (136 mg, 30 μmol) in DCM and Et₃N (110 μL). The resulting mixturewas stirred for 10 minutes. The solvent was evaporated and the residuewas dissolved in AcOH and purified by preparative HPLC to yield thetitle compound (2.1 mg). MS m/z [M+H]⁺calc'd for C₂₂H₂₆ClNO₅, 420.15.found 420.2.

Example 12

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, thefollowing compound was prepared as the parent compound. These compounds,where X is —CH(R¹⁵)—OR¹⁶, are depicted by formula Ve:

R¹, R⁴, and R¹⁵═H; R⁵═Cl

MS m/z: [M + H]⁺ Ex. R¹⁶ Formula calcd found 1 benzyl C₂₈H₃₀ClNO₅ 496.18496.2

-   1.    (2S,4R)-4-(2-Benzyloxyacetylamino)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-pentanoic    acid

Example 13(2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-formylamino-2-hydroxymethyl-2-methyl-pentanoicAcid

Formic acid (3 mg, 32 μmol) was combined with HATU (12 mg, 32 μmol) andDMF (0.2 mL) and stirred for 5 minutes. DIPEA (17 μl, 96 μmol) wasadded, followed by(2S,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-pentanoicacid (79 mg, 38 μmol) pre-dissolved in DMF (0.5 mL) and DIPEA (17 μl, 96μmol). The resulting mixture was stirred for 15 minutes. The solvent wasremoved in vacuo and the residue was dissolved in AcOH and purified bypreparative HPLC to yield the title compound (0.5 mg). MS m/z[M+H]⁺calc'd for C₂₀H₂₂ClNO₄, 376.12. found 376.2.

Assay 1 In Vitro Assays for the Quantitation of Inhibitor Potencies atHuman and Rat NEP, and Human ACE

The inhibitory activities of compounds at human and rat neprilysin (EC3.4.24.11; NEP) and human angiotensin converting enzyme (ACE) weredetermined using in vitro assays as described below.

Extraction of NEP Activity from Rat Kidneys

Rat NEP was prepared from the kidneys of adult Sprague Dawley rats.Whole kidneys were washed in cold phosphate buffered saline (PBS) andbrought up in ice-cold lysis buffer (1% Triton X-114, 150 mM NaCl, 50 mMtris(hydroxymethyl)aminomethane (Tris) pH 7.5; Bordier (1981) J. Biol.Chem. 256: 1604-1607) in a ratio of 5 mL of buffer for every gram ofkidney. Samples were homogenized on ice using a polytron hand heldtissue grinder. Homogenates were centrifuged at 1000×g in a swingingbucket rotor for 5 minutes at 3° C. The pellet was resuspended in 20 mLof ice cold lysis buffer and incubated on ice for 30 minutes. Samples(15-20 mL) were then layered onto 25 mL of ice-cold cushion buffer (6%w/v sucrose, 50 mM pH 7.5 Tris, 150 mM NaCl, 0.06%, Triton X-114),heated to 37° C. for 3-5 minutes and centrifuged at 1000×g in a swingingbucket rotor at room temperature for 3 minutes. The two upper layerswere aspirated off, leaving a viscous oily precipitate containing theenriched membrane fraction. Glycerol was added to a concentration of 50%and samples were stored at −20° C. Protein concentrations werequantitated using a BCA detection system with bovine serum albumin (BSA)as a standard.

Enzyme Inhibition Assays

Recombinant human NEP and recombinant human ACE were obtainedcommercially (R&D Systems, Minneapolis, Minn., catalog numbers 1182-ZNand 929-ZN, respectively). The fluorogenic peptide substrateMca-D-Arg-Arg-Leu-Dap-(Dnp)-OH (Medeiros et al. (1997) Braz. J. Med.Biol. Res. 30:1157-62; Anaspec, San Jose, Calif.) andAbz-Phe-Arg-Lys(Dnp)-Pro-OH (Araujo et al. (2000) Biochemistry39:8519-8525; Bachem, Torrance, Calif.) were used in the NEP and ACEassays respectively.

The assays were performed in 384-well white opaque plates at 37° C.using the fluorogenic peptide substrates at a concentration of 10 μM inAssay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethyleneglycol sorbitan monolaurate (Tween-20), 10 μM ZnSO₄; ACE: 50 mM HEPES,pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO₄). The respective enzymeswere used at concentrations that resulted in quantitative proteolysis of1 μM of substrate after 20 minutes at 37° C.

Test compounds were assayed over the range of concentrations from 10 μMto 20 pM. Test compounds were added to the enzymes and incubated for 30minute at 37° C. prior to initiating the reaction by the addition ofsubstrate. Reactions were terminated after 20 minutes of incubation at37° C. by the addition of glacial acetic acid to a final concentrationof 3.6% (v/v).

Plates were read on a fluorometer with excitation and emissionwavelengths set to 320 nm and 405 nm, respectively Inhibition constantswere obtained by nonlinear regression of the data using the equation(GraphPad Software, Inc., San Diego, Calif.):

v=v ₀/[1+(I/K′)]

where v is the reaction rate, v₀ is the uninhibited reaction rate, I isthe inhibitor concentration and K′ is the apparent inhibition constant.

Compounds of the invention were tested in this assay and found to havepK_(i) values at human NEP as follows. In general, either the prodrugcompounds did not inhibit the enzyme in this in vitro assay, or theprodrugs were not tested (n.d.) since activity would not be expected.

Ex. pK_(i)  1 8.0-9.0 2-1 8.0-9.0 2-2 8.0-9.0  3 ≧9.0 4-1 7.5-8.0 4-27.5-8.0 4-3 7.5-8.0 4-4 8.0-9.0 4-5 7.5-8.0 4-6 8.0-9.0 4-7 8.0-9.0 4-87.5-8.0 4-9 8.0-9.0  4-10 7.5-8.0  4-11 7.5-8.0  4-12 8.0-9.0  4-138.0-9.0  4-14 7.5-8.0  4-15 8.0-9.0  4-16 8.0-9.0  4-17 7.5-8.0  4-18≧9.0  4-19 7.5-8.0  4-20 ≧9.0  4-21 8.0-9.0  4-22 8.0-9.0  4-23 7.5-8.0 4-24 8.0-9.0  4-25 ≧9.0  4-26 ≧9.0  4-27 8.0-9.0  4-28 8.0-9.0  4-297.5-8.0  4-30 7.5-8.0  4-31 7.5-8.0  4-32 7.5-8.0  4-33 8.0-9.0  4-347.5-8.0  4-35 7.5-8.0  4-36 8.0-9.0  4-37 7.5-8.0  4-38 7.5-8.0  4-397.5-8.0  4-40 7.5-8.0  4-41 8.0-9.0  4-42 7.5-8.0  4-43 8.0-9.0  4-448.0-9.0  4-45 8.0-9.0  4-46 8.0-9.0  4-47 8.0-9.0  4-48 8.0-9.0  4-498.0-9.0  4-50 7.5-8.0  5 ≧9.0 6-1 7.5-8.0 6-2 7.5-8.0 6-3 8.0-9.0 6-48.0-9.0 6-5 8.0-9.0 6-6 7.5-8.0 6-7 8.0-9.0 6-8 8.0-9.0 6-9 8.0-9.0 6-10 8.0-9.0  6-11 8.0-9.0  6-12 8.0-9.0  6-13 8.0-9.0  6-14 8.0-9.0 6-15 8.0-9.0  6-16 8.0-9.0  6-17 8.0-9.0  7 ≧9.0 8-1 7.5-8.0 8-28.0-9.0 8-3 8.0-9.0 8-4 8.0-9.0 8-5 8.0-9.0 8-6 8.0-9.0  9 8.0-9.0 10-1 8.0-9.0 10-2  8.0-9.0 10-3  7.5-8.0 10-4  7.5-8.0 10-5  8.0-9.0 10-6 7.5-8.0 10-7  7.5-8.0 10-8  7.5-8.0 10-9  7.5-8.0 11 8.0-9.0 12 7.5-8.013 8.0-9.0

Assay 2 Pharmacodynamic (PD) Assay for ACE and NEP Activity inAnesthetized Rats

Male, Sprague Dawley, normotensive rats are anesthetized with 120 mg/kg(i.p.) of inactin. Once anesthetized, the jugular vein, carotid artery(PE 50 tubing) and bladder (flared PE 50 tubing) catheters arecannulated and a tracheotomy is performed (Teflon Needle, size 14 gauge)to facilitate spontaneous respiration. The animals are then allowed a 60minute stabilization period and kept continuously infused with 5 mL/kg/hof saline (0.9%) throughout, to keep them hydrated and ensure urineproduction. Body temperature is maintained throughout the experiment byuse of a heating pad. At the end of the 60 minute stabilization period,the animals are dosed intravenously (i.v.) with two doses of AngI (1.0μg/kg, for ACE inhibitor activity) at 15 minutes apart. At 15 minutespost-second dose of AngI, the animals are treated with vehicle or testcompound. Five minutes later, the animals are additionally treated witha bolus i.v. injection of atrial natriuretic peptide (ANP; 30 μg/kg).Urine collection (into pre-weighted eppendorf tubes) is startedimmediately after the ANP treatment and continued for 60 minutes. At 30and 60 minutes into urine collection, the animals are re-challenged withAngI. Blood pressure measurements are done using the Notocord system(Kalamazoo, Mich.). Urine samples are frozen at −20° C. until used forthe cGMP assay. Urine cGMP concentrations are determined by EnzymeImmuno Assay using a commercial kit (Assay Designs, Ann Arbor, Mich.,Cat. No. 901-013). Urine volume is determined gravimetrically. UrinarycGMP output is calculated as the product of urine output and urine cGMPconcentration. ACE inhibition is assessed by quantifying the %inhibition of pressor response to AngI. NEP inhibition is assessed byquantifying the potentiation of ANP-induced elevation in urinary cGMPoutput.

Assay 3 In Vivo Evaluation of Antihypertensive Effects in the ConsciousSHR Model of Hypertension

Spontaneously hypertensive rats (SHR, 14-20 weeks of age) are allowed aminimum of 48 hours acclimation upon arrival at the testing site withfree access to food and water. For blood pressure recording, theseanimals are surgically implanted with small rodent radiotransmitters(telemetry unit; DSI Models TA11PA-C40 or C50-PXT, Data Science Inc.,USA). The tip of the catheter connected to the transmitter is insertedinto the descending aorta above the iliac bifurcation and secured inplace with tissue adhesive. The transmitter is kept intraperitoneallyand secured to the abdominal wall while closing of the abdominalincision with a non-absorbable suture. The outer skin is closed withsuture and staples. The animals are allowed to recover with appropriatepost-operative care. On the day of the experiment, the animals in theircages are placed on top of the telemetry receiver units to acclimate tothe testing environment and baseline recording. After at least of 2hours baseline measurement is taken, the animals are then dosed withvehicle or test compound and followed out to 24 hours post-dose bloodpressure measurement. Data is recorded continuously for the duration ofthe study using Notocord software (Kalamazoo, Mich.) and stored aselectronic digital signals. Parameters measured are blood pressure(systolic, diastolic and mean arterial pressure) and heart rate.

Assay 4 In Vivo Evaluation of Antihypertensive Effects in the ConsciousDOCA-Salt Rat Model of Hypertension

CD rats (male, adult, 200-300 grams, Charles River Laboratory, USA) areallowed a minimum of 48 hours acclimation upon arrival at the testingsite before they are placed on a high salt diet. One week after thestart of the high salt diet (8% in food or 1% NaCl in drinking water), adeoxycorticosterone acetate (DOCA) pellet (100 mg, 90 days release time,Innovative Research of America, Sarasota, Fla.) is implantedsubcutaneously and unilateral nephrectomy is performed. At this time,the animals are also surgically implanted with small rodentradiotransmitters for blood pressure measurement (see Assay 3 fordetails). The animals are allowed to recover with appropriatepost-operative care. Study design, data recording, and parametersmeasured is similar to that described for Assay 3.

Assay 5 In Vivo Evaluation of Antihypertensive Effects in the ConsciousDahl/SS Rat Model of Hypertension

Male, Dahl salt sensitive rats (Dahl/SS, 6-7 weeks of age from CharlesRiver Laboratory, USA) are allowed at least 48 hours of acclimation uponarrival at the testing site before they were placed on a 8% NaCl highsalt diet (TD.92012, Harlan, USA) then surgically implanted with smallrodent radiotransmitters for blood pressure measurement (see Assay 3 fordetails). The animals are allowed to recover with appropriatepost-operative care. At approximately 4 to 5 weeks from the start ofhigh salt diet, these animals are expected to become hypertensive. Oncethe hypertension level is confirmed, these animals are used for thestudy while continued with the high salt diet to maintain theirhypertension level. Study design, data recording, and parametersmeasured is similar to that described in Assay 3.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

1. (canceled)
 2. The compound of claim 22, where R¹ is H.
 3. Thecompound of claim 22, where R¹ is selected from the group consisting ofC₁₋₈alkyl optionally substituted with one or more fluoro atoms;C₁₋₃alkylene-C₆₋₁₀aryl; C₁₋₃alkylene-C₁₋₉heteroaryl; C₃₋₇cycloalkyl;C₂₋₃alkylene-OH; —[(CH₂)₂O]₁₋₃CH₃; C₁₋₆alkylene-OC(O)R²⁰;C₁₋₆alkylene-NR²¹R²²; —CH₂CH(NH₂)—COOCH₃; C₁₋₆alkylene-C(O)R²³; C₀₋₆alkylenemorpholine; C₁₋₆ alkylene-SO₂—C₁₋₆alkyl;

4-7. (canceled)
 8. The compound of claim 22, where R⁴ is hydrogen orhalo.
 9. The compound of claim 22, where R⁵ is halo. 10-21. (canceled)22. A compound of formula e:

where: R¹ is selected from the group consisting of H; C₁₋₈alkyloptionally substituted with one or more fluoro atoms;C₁₋₃alkylene-C₆₋₁₀aryl; C₁₋₃alkylene-C₁₋₉heteroaryl; C₃₋₇cycloalkyl;C₂₋₃alkylene-OH; —[(CH₂)₂O]₁₋₃CH₃; C₁₋₆alkylene-OC(O)R²⁰;C₁₋₆alkylene-NR²¹R²²; —CH₂CH(NH₂)—COOCH₃; C₁₋₆alkylene-C(O)R²³;C₀₋₆alkylenemorpholine; C₁₋₆alkylene-SO₂—C₁₋₆alkyl;

where R²⁰ is selected from the group consisting of C₁₋₆alkyl,—O—C₁₋₆alkyl, C₃₋₇cycloalkyl, —O—C₃₋₇cycloalkyl, phenyl, —O-phenyl,—NR²¹R²², —CH(R²⁵)—NH₂, —CH(R²⁵)—NHC(O)O—C₁₋₆alkyl, and—CH(NH₂)CH₂COOCH₃; and R²¹ and R²² are independently selected from thegroup consisting of H, C₁₋₆alkyl, and benzyl; or R²¹ and R²² are takentogether as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—; R²³ isselected from the group consisting of —O—C₁₋₆alkyl, —O-benzyl, and—NR²¹R²²; R²⁴ is C₁₋₆alkyl or C₀₋₆alkylene-C₆₋₁₀aryl; and R²⁵ is H,—CH₃, —CH(CH₃)₂, phenyl, or benzyl; R² is —OH, —CH₂OH, or—CH₂—O—C₁₋₆alkyl; and R³ is H or —CH₃; R⁴ and R⁵ are independentlyselected from the group consisting of hydrogen, halo, —OH, —CH₃—OCH₃,—CN, and —CF₃; R¹⁵ H or C₁₋₆alkyl; and R¹⁶ is H, C₁₋₆alkyl,—[(CH₂)₂O]₁₋₃CH₃, C₁₋₉heteroaryl, benzyl, or C₆₋₁₀aryl optionallysubstituted with —OH or —OCH₃; or R¹⁵ and R¹⁶ are taken together to form—(CH₂)₄; or a pharmaceutically acceptable salt thereof.
 23. The compoundof claim 22 where R² is —OH and R³ is H or R² is —CH₂OH and R³ is —CH₃.24. The compound of claim 22, where R¹⁵ is H; R¹⁶ is selected from H,—CH₃, —CH(CH₃)₂, —CH₂CH₃, pyridine, benzyl, phenyl, phenyl substitutedwith —OH, and phenyl substituted with —OCH₃; or R¹⁵ and R¹⁶ are takentogether to form —(CH₂)₄—. 25-27. (canceled)
 28. A pharmaceuticalcomposition comprising the compound of claim 22 and a pharmaceuticallyacceptable carrier.
 29. (canceled)
 30. The pharmaceutical composition ofclaim 28, further comprising an AT₁ receptor antagonist.
 31. A methodfor treating hypertension, heart failure, or renal disease, comprisingadministering to a patient a therapeutically effective amount of thecompound of claim 22.